Vector system

ABSTRACT

The present invention provides a vector system comprising a nucleotide sequence coding for an antibody. In particular, the present invention relates to the use of such a vector system in a subject, where the nucleotide sequence is expressed in vivo to produce said antibody.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a Divisional of Ser. No. 10/060,585 filed 29 Jan.2002 which claims priority to Continuation-in-Part application of U.S.patent application Ser. No. 09/445,375 filed 21 Mar. 2000 as the 35U.S.C. § 371(c) date and is the National Phase of PCT/GB98/01627 filed 4Jun. 1998, which claims priority under 35 U.S.C. § 119 to Great Britainpatent application number 9711579.4 filed 4 Jun. 1997, Great Britainpatent application number 9713150.2 filed 20 Jun. 1997 and Great Britainpatent application number 9714230.1 filed 4 Jul. 1997, and aContinuation-in-Part application of PCT No. PCT/GB00/04137, designatinginter alia the US and published in English, filed 13 Nov. 2000, claimingpriority from PCT/GB99/03859 and GB 0003527.9 and GB 0005071.6.

Reference is made in this application to the following applications:

U.S. patent application Ser. No. 09/532,909 filed 22 Mar. 2000 claimingpriority from PCT/GB98/02867 and GB 0005846.1

U.S. patent application Ser. No. 09/533,276 filed 22 Mar. 2000 claimingpriority from PCT/GB98/02867 and GB 0005841.2

U.S. patent application Ser. No. 09/533,295 filed 22 Mar. 2000 claimingpriority from PCT/GB98/02867 and GB 0005844.6

PCT No. PCT/GB98/02867 filed 23 Sep. 1998 claiming priority of GB9720465.5 designating inter alia the US;

PCT No. PCT/GB98/02885 filed 23 Sep. 1998 claiming priority of GB9720216.2 designating inter alia the US;

U.S. Ser. No. 09/238,356 filed 27 Jan. 1999 claiming priority of GB9811037.2 and GB 9727135.7;

PCT No. PCT/GB99/00325 filed 17 Feb. 1999 claiming priority of GB9803351.7 designating inter alia the US;

PCT No. PCT/GB99/00672 filed 5 May 1999 claiming priority of GB9902081.0, GB 9818103.5 and GB 9804841.6 designating inter alia the US;

PCT No. PCT/GB99/00764 filed 5 May 1999 claiming priority of GB9902081.0, GB 9818103.5 and GB 9804841.6 designating interalia the US;

U.S. 60/093,149 filed 17 Jul. 1998 claiming priority of GB 9811152.9;

PCT No. PCT/GB99/03866 filed 19 Nov. 1999 claiming priority of GB9825524.3 designating inter alia the US;

PCT No. PCT/GB00/00520 filed 15 Feb. 2000 claiming priority of GB9903408.4 designating inter alia the US; and

PCT No. PCT/GB99/03181 filed 22 Sep. 1999 claiming priority of GB9903538.8, GB 9901906.9 and PCT/GB98/02885 designating inter alia theUS.

Each of the aforementioned applications, and each document cited in eachof the aforementioned applications—either in the text thereof or duringthe prosecution thereof—(“application cited documents”) and eachdocument referenced or cited in each of the application cited documents,is hereby incorporated herein by reference. Likewise, each documentcited in this text (“herein cited documents”) and each documentreferenced or cited in herein cited documents, is hereby incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to a vector system.

In particular, the present invention relates to a vector systemcomprising a nucleotide sequence capable of encoding an antibody invivo.

SUMMARY OF ASPECTS OF THE PRESENT INVENTION

According to a first aspect of the present invention there is provided avector system comprising a nucleotide sequence (“NS”) coding for anantibody.

The vector system may be a non-viral system or a viral vector system.

The vector system may also comprise a nucleotide sequence of interest(“NOI”) which optionally encodes a protein of interest (“POI”).

According to a second aspect of the present invention there is provideda method of treating and/or preventing a disease in a subject in need ofsame, the method comprising the step of administering a vector systemaccording to the first aspect of the invention to the subject, such thatthe NS is expressed in vivo to produce said antibody.

According to a third aspect, the present invention also provides the useof a vector system according to the first aspect of the invention in themanufacture of a medicament to treat and/or prevent a disease in asubject in need of same, wherein the NS is expressed in vivo to producesaid antibody.

The disease may be a cancerous or non-cancerous disease.

For cancerous diseases, the present invention also provides a method ofdelivering a nucleotide sequence of interest (“NOI”) and/or a product ofinterest (“POI”) to a tumour, which comprises the step of using a vectorsystem according to the first aspect of the invention wherein theantibody is a tumour-interacting protein (“TIP”).

DETAILED DESCRIPTION OF THE INVENTION Vector System

The present invention relates to a vector system, in particular a vectorsystem comprising a nucleotide sequence coding for an antibody.

As it is well known in the art, a vector is a tool that allows orfacilitates the transfer of an entity from one environment to another.By way of example, some vectors used in recombinant DNA techniques allowentities—such as a segment of DNA (such as a heterologous DNA segment,such as a heterologous cDNA segment)—to be transferred into a targetcell. Optionally, once within the target cell, the vector may then serveto maintain the heterologous DNA within the cell or may act as a unit ofDNA replication. Examples of vectors used in recombinant DNA techniquesinclude plasmids, chromosomes, artificial chromosomes or viruses.

Gene therapy includes any one or more of: the addition, the replacement,the deletion, the supplementation, the manipulation etc. of one or morenucleotide sequences in, for example, one or more targeted sites—such astargeted cells. If the targeted sites are targeted cells, then the cellsmay be part of a tissue or an organ. General teachings on gene therapymay be found in Molecular Biology (Ed Robert Meyers, Pub VCH, such aspages 556-558).

By way of further example, gene therapy also provides a means by whichany one or more of: a nucleotide sequence, such as a gene, can beapplied to replace or supplement a defective gene; a pathogenic gene orgene product can be eliminated; a new gene can be added in order, forexample, to create a more favourable phenotype; cells can be manipulatedat the molecular level to treat cancer (Schmidt-Wolf and Schmidt-Wolf,1994, Annals of Hematology 69; 273-279) or other conditions—such asimmune, cardiovascular, neurological, inflammatory or infectiousdisorders; antigens can be manipulated and/or introduced to elicit animmune response—such as genetic vaccination.

The vector of the present invention may be a viral vector or a non-viralvector.

In a first preferred embodiment, the vector is a non-viral vector.Non-viral delivery systems include but are not limited to DNAtransfection methods. Here transfection includes a process using anon-viral vector to deliver a gene to a target mammalian cell. Typicaltransfection methods include electroporation, DNA biolistics,lipid-mediated transfection, compacted DNA-mediated transfection,liposomes, immunoliposomes, lipofectin, cationic agent-mediated,cationic facial amphiphiles (CFAs) (Nature Biotechnology 1996 14; 556),and combinations thereof. Viral delivery systems include but are notlimited to adenovirus vector, an adeno-associated viral (AAV) vector, aherpes viral vector, retroviral vector, lentiviral vector, baculoviralvector. Other examples of vectors include ex vivo delivery systems—whichinclude but are not limited to DNA transfection methods such aselectroporation, DNA biolistics, lipid-mediated transfection, compactedDNA-mediated transfection).

In a second preferred embodiment the vector is a viral vector.

Preferably the vector is a retroviral vector.

In recent years, retroviruses have been proposed for use in genetherapy. Essentially, retroviruses are RNA viruses with a life cycledifferent to that of lytic viruses. In this regard, when a retrovirusinfects a cell, its genome is converted to a DNA form. In slightly moredetail, a retrovirus is a virus which contains genomic RNA which onentry into a host cell is converted to a DNA molecule by a reversetranscriptase enzyme. The DNA copy serves as a template for theproduction of new RNA genomes and virally encoded proteins necessary forthe assembly of infectious viral particles. Thus, a retrovirus is aninfectious entity that replicates through a DNA intermediate.

There are many retroviruses and examples include: murine leukemia virus(MLV), human immunodeficiency virus (HIV), equine infectious anaemiavirus (EIAV), mouse mammary tumour virus (MMTV), Rous sarcoma virus(RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus(Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murinesarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), Avianmyelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus(AEV).

A detailed list of retroviruses may be found in Coffin et al(“Retroviruses” 1997 Cold Spring Harbour Laboratory Press Eds: J MCoffin, S M Hughes, H E Varmus pp 758-763).

Details on the genomic structure of some retroviruses may be found inthe art. By way of example, details on HIV may be found from the NCBIGenbank (i.e. Genome Accession No. AF033819).

All retroviruses contain three major coding domains, gag, pol, env,which code for essential virion proteins. Nevertheless, retroviruses maybe broadly divided into two categories: namely, “simple” and “complex”.These categories are distinguishable by the organisation of theirgenomes. Simple retroviruses usually carry only this elementaryinformation. In contrast, complex retroviruses also code for additionalregulatory proteins derived from multiple spliced messages.

Retroviruses may even be further divided into seven groups. Five ofthese groups represent retroviruses with oncogenic potential. Theremaining two groups are the lentiviruses and the spumaviruses. A reviewof these retroviruses is presented in “Retroviruses” (1997 Cold SpringHarbour Laboratory Press Eds: J M Coffin, S M Hughes, H E Varmus pp1-25).

All oncogenic members except the human T-cell leukemia virus-bovineleukemia virus group (HTLV-BLV) are simple retroviruses. HTLV, BLV andthe lentiviruses and spumaviruses are complex. Some of the best studiedoncogenic retroviruses are Rous sarcoma virus (RSV), mouse mammarytumour virus (MMTV) and murine leukemia virus (MLV) and the human T-cellleukemia virus (HTLV).

The vector system of the present invention may be a lentiviral vectorsystem.

The lentivirus group of retroviruses can be split into “primate” and“non-primate”. Examples of primate lentiviruses include the humanimmunodeficiency virus (HIV), the causative agent of humanauto-immunodeficiency syndrome (AIDS), and the simian immunodeficiencyvirus (SIV). The non-primate lentiviral group includes the prototype“slow virus” visna/maedi virus (VMV), as well as the related caprinearthritis-encephalitis virus (CAEV), equine infectious anaemia virus(EIAV) and the more recently described feline immunodeficiency virus(FIV) and bovine immunodeficiency virus (BIV).

A distinction between the lentivirus family and other types ofretroviruses is that lentiviruses have the capability to infect bothdividing and non-dividing cells (Lewis et al 1992 EMBO. J 11; 3053-3058,Lewis and Emerman 1994 J. Virol. 68: 510-516). In contrast, otherretroviruses—such as MLV—are unable to infect non-dividing cells such asthose that make up, for example, muscle, brain, lung and liver tissue.

In one embodiment of the present invention, the features of adenovirusesmay be combined with the genetic stability of retroviruses/lentiviruseswhich can be used to transduce target cells to become transientretroviral producer cells capable of stably infect neighbouring cells.Such retroviral producer cells which are engineered to express anantibody can be implanted in organisms such as animals or humans.

Preferred vectors for use in accordance with the present invention arerecombinant pox viral vectors such as fowl pox virus (FPV), entomopoxvirus, vaccinia virus such as NYVAC, canarypox virus, MVA or othernon-replicating viral vector systems, such as those described forexample in WO 95/30018.

The present invention also provides a hybrid viral vector system for invivo delivery of a nucleotide sequence encoding an antibody, whichsystem comprises one or more primary viral vectors which encode asecondary viral vector, the primary vector or vectors capable ofinfecting a first target cell and of expressing therein the secondaryviral vector, which secondary vector is capable of transducing asecondary target cell.

Preferably the primary vector is obtainable from or is based on anadenoviral vector and/or the secondary viral vector is obtainable fromor is based on a retroviral vector preferably a lentiviral vector.

During the process of infection, a retrovirus initially attaches to aspecific cell surface receptor. On entry into the susceptible host cell,the retroviral RNA genome is then copied to DNA by the virally encodedreverse transcriptase which is carried inside the parent virus. This DNAis transported to the host cell nucleus where it subsequently integratesinto the host genome. At this stage, it is typically referred to as theprovirus. The provirus is stable in the host chromosome during celldivision and is transcribed like other cellular proteins. The provirusencodes the proteins and packaging machinery required to make morevirus, which can leave the cell by a process sometimes called “budding”.

As already indicated, each retroviral genome comprises genes called gag,pol and env which code for virion proteins and enzymes. These genes areflanked at both ends by regions called long terminal repeats (LTRs). TheLTRs are responsible for proviral integration, and transcription. Theyalso serve as enhancer-promoter sequences. In other words, the LTRs cancontrol the expression of the viral gene. Encapsidation of theretroviral RNAs occurs by virtue of a psi sequence located at the 5′ endof the viral genome.

The LTRs themselves are identical sequences that can be divided intothree elements, which are called U3, R and U5. U3 is derived from thesequence unique to the 3′ end of the RNA. R is derived from a sequencerepeated at both ends of the RNA and U5 is derived from the sequenceunique to the 5′ end of the RNA. The sizes of the three elements canvary considerably among different retroviruses.

For ease of understanding, a simple, generic diagram (not to scale) of aretroviral genome showing the elementary features of the LTRs, gag, poland env is presented below.

For the viral genome, the site of transcription initiation is at theboundary between U3 and R in the left hand side LTR (as shown above) andthe site of poly (A) addition (termination) is at the boundary between Rand U5 in the right hand side LTR (as shown above). U3 contains most ofthe transcriptional control elements of the provirus, which include thepromoter and multiple enhancer sequences responsive to cellular and insome cases, viral transcriptional activator proteins. Some retroviruseshave any one or more of the following genes that code for proteins thatare involved in the regulation of gene expression: tat, rev, tax andrex.

As shown in the diagram above, the basic molecular organisation of aretroviral RNA genome is (5′) R-U5-gag, pol, env-U3-R (3′). In aretroviral vector genome gag, pol and env are absent or not functional.The R regions at both ends of the RNA are repeated sequences. U5 and U3represent sequences unique, respectively, to the 5′ and 3′ ends of theRNA genome. These three sets of end sequences go to form the longterminal repeats (LTRs) in the proviral DNA, which is the form of thegenome which integrates into the genome of the infected cell. The LTRsin a wild type retrovirus consist of (5′)U3-R-U5 (3′), and thus U3 andU5 both contain sequences which are important for proviral integration.Other essential sequences required in the genome for proper functioninginclude a primer binding site for first strand reverse transcription, aprimer binding site for second strand reverse transcription and apackaging signal.

With regard to the structural genes gag, pol and env themselves and inslightly more detail, gag encodes the internal structural protein of thevirus. Gag is proteolytically processed into the mature proteins MA(matrix), CA (capsid), NC (nucleocapsid). The gene pol encodes thereverse transcriptase (RT), which contains both DNA polymerase, andassociated RNase H activities and integrase (IN), which mediatesreplication of the genome. The gene env encodes the surface (SU)glycoprotein and the transmembrane (TM) protein of the virion, whichform a complex that interacts specifically with cellular receptorproteins. This interaction leads ultimately to fusion of the viralmembrane with the cell membrane.

The envelope protein is a viral protein which coats the viral particleand plays an essential role in permitting viral entry into a targetcell. The envelope glycoprotein complex of retroviruses includes twopolypeptides: an external, glycosylated hydrophilic polypeptide (SU) anda membrane-spanning protein (TM). Together, these form an oligomeric“knob” or “knobbed spike” on the surface of a virion. Both polypeptidesare encoded by the env gene and are synthesised in the form of apolyprotein precursor that is proteolytically cleaved during itstransport to the cell surface. Although uncleaved Env proteins are ableto bind to the receptor, the cleavage event itself is necessary toactivate the fusion potential of the protein, which is necessary forentry of the virus into the host cell. Typically, both SU and TMproteins are glycosylated at multiple sites. However, in some viruses,exemplified by MLV, TM is not glycosylated.

Although the SU and TM proteins are not always required for the assemblyof enveloped virion particles as such, they do play an essential role inthe entry process. In this regard, the SU domain binds to a receptormolecule—often a specific receptor molecule—on the target cell. It isbelieved that this binding event activates the membrane fusion-inducingpotential of the TM protein after which the viral and cell membranesfuse. In some viruses, notably MLV, a cleavage event—resulting in theremoval of a short portion of the cytoplasmic tail of TM—is thought toplay a key role in uncovering the full fusion activity of the protein(Brody et al 1994 J. Virol. 68: 4620-4627, Rein et al 1994 J. Virol. 68:1773-1781). This cytoplasmic “tail”, distal to the membrane-spanningsegment of TM remains on the internal side of the viral membrane and itvaries considerably in length in different retroviruses.

Thus, the specificity of the SU/receptor interaction can define the hostrange and tissue tropism of a retrovirus. In some cases, thisspecificity may restrict the transduction potential of a recombinantretroviral vector. Here, transduction includes a process of using aviral vector to deliver a non-viral gene to a target cell. For thisreason, many gene therapy experiments have used MLV. A particular MLVthat has an envelope protein called 4070A is known as an amphotropicvirus, and this can also infect human cells because its envelope protein“docks” with a phosphate transport protein that is conserved between manand mouse. This transporter is ubiquitous and so these viruses arecapable of infecting many cell types. In some cases however, it may bebeneficial, especially from a safety point of view, to specificallytarget restricted cells. To this end, several groups have engineered amouse ecotropic retrovirus, which unlike its amphotropic relativenormally only infects mouse cells, to specifically infect particularhuman cells. Replacement of a fragment of an envelope protein with anerythropoietin segement produced a recombinant retrovirus which thenbound specifically to human cells that expressed the erythropoietinreceptor on their surface, such as red blood cell precursors (Maulik andPatel 1997 “Molecular Biotechnology: Therapeutic Applications andStrategies” 1997. Wiley-Liss Inc. pp 45.).

In addition to gag, pol and env, the complex retroviruses also contain“accessory” genes which code for accessory or auxiliary proteins.Accessory or auxiliary proteins are defined as those proteins encoded bythe accessory genes in addition to those encoded by the usualreplicative or structural genes, gag, pol and env. These accessoryproteins are distinct from those involved in the regulation of geneexpression, like those encoded by tat, rev, tax and rex. Examples ofaccessory genes include one or more of vif, vpr, vpx, vpu and nef. Theseaccessory genes can be found in, for example, HIV (see, for examplepages 802 and 803 of “Retroviruses” Ed. Coffin et al Pub. CSHL 1997).Non-essential accessory proteins may function in specialised cell types,providing functions that are at least in part duplicative of a functionprovided by a cellular protein. Typically, the accessory genes arelocated between pol and env, just downstream from env including the U3region of the LTR or overlapping portions of the env and each other.

The complex retroviruses have evolved regulatory mechanisms that employvirally encoded transcriptional activators as well as cellulartranscriptional factors. These trans-acting viral proteins serve asactivators of RNA transcription directed by the LTRs. Thetranscriptional trans-activators of the lentiviruses are encoded by theviral tat genes. Tat binds to a stable, stem-loop, RNA secondarystructure, referred to as TAR, one function of which is to apparentlyoptimally position Tat to trans-activate transcription.

As mentioned earlier, retroviruses have been proposed as a deliverysystem (other wise expressed as a delivery vehicle or delivery vector)for inter alia the transfer of a NOI, or a plurality of NOIs, to one ormore sites of interest. The transfer can occur in vitro, ex vivo, invivo, or combinations thereof. When used in this fashion, theretroviruses are typically called retroviral vectors or recombinantretroviral vectors. Retroviral vectors have even been exploited to studyvarious aspects of the retrovirus life cycle, including receptor usage,reverse transcription and RNA packaging (reviewed by Miller, 1992 CurrTop Microbiol Immunol 158:1-24).

In a typical recombinant retroviral vector for use in gene therapy, atleast part of one or more of the gag, pol and env protein coding regionsmay be removed from the virus. This makes the retroviral vectorreplication-defective. The removed portions may even be replaced by aNOI in order to generate a virus capable of integrating its genome intoa host genome but wherein the modified viral genome is unable topropagate itself due to a lack of structural proteins. When integratedin the host genome, expression of the NOI occurs—resulting in, forexample, a therapeutic effect. Thus, the transfer of a NOI into a siteof interest is typically achieved by: integrating the NOI into therecombinant viral vector; packaging the modified viral vector into avirion coat; and allowing transduction of a site of interest—such as atargeted cell or a targeted cell population.

It is possible to propagate and isolate quantities of retroviral vectors(e.g. to prepare suitable titres of the retroviral vector) forsubsequent transduction of, for example, a site of interest by using acombination of a packaging or helper cell line and a recombinant vector.

In some instances, propagation and isolation may entail isolation of theretroviral gag, pol and env genes and their separate introduction into ahost cell to produce a “packaging cell line”. The packaging cell lineproduces the proteins required for packaging retroviral DNA but itcannot bring about encapsidation due to the lack of a psi region.However, when a recombinant vector carrying a NOI and a psi region isintroduced into the packaging cell line, the helper proteins can packagethe psi-positive recombinant vector to produce the recombinant virusstock. This can be used to infect cells to introduce the NOI into thegenome of the cells. The recombinant virus whose genome lacks all genesrequired to make viral proteins can infect only once and cannotpropagate. Hence, the NOI is introduced into the host cell genomewithout the generation of potentially harmful retrovirus. A summary ofthe available packaging lines is presented in “Retroviruses” (1997 ColdSpring Harbour Laboratory Press Eds: J M Coffin, S M Hughes, H E Varmuspp 449). However, this technique can be problematic in the sense thatthe titre levels are not always at a satisfactory level. Nevertheless,the design of retroviral packaging cell lines has evolved to address theproblem of inter alia the spontaneous production of helper virus thatwas frequently encountered with early designs. As recombination isgreatly facilitated by homology, reducing or eliminating homologybetween the genomes of the vector and the helper has reduced the problemof helper virus production.

More recently, packaging cells have been developed in which the gag, poland env viral coding regions are carried on separate expression plasmidsthat are independently transfected into a packaging cell line so thatthree recombinant events are required for wild type viral production.This strategy is sometimes referred to as the three plasmid transfectionmethod (Soneoka et al 1995 Nucl. Acids Res. 23: 628-633).

Transient transfection can also be used to measure vector productionwhen vectors are being developed. In this regard, transient transfectionavoids the longer time required to generate stable vector-producing celllines and is used if the vector or retroviral packaging components aretoxic to cells. Components typically used to generate retroviral vectorsinclude a plasmid encoding the Gag/Pol proteins, a plasmid encoding theEnv protein and a plasmid containing a NOI. Vector production involvestransient transfection of one or more of these components into cellscontaining the other required components. If the vector encodes toxicgenes or genes that interfere with the replication of the host cell,such as inhibitors of the cell cycle or genes that induce apotosis, itmay be difficult to generate stable vector-producing cell lines, buttransient transfection can be used to produce the vector before thecells die. Also, cell lines have been developed using transientinfection that produce vector titre levels that are comparable to thelevels obtained from stable vector-producing cell lines (Pear et a/1993,PNAS 90:8392-8396).

In view of the toxicity of some HIV proteins—which can make it difficultto generate stable HIV-based packaging cells —HIV vectors are usuallymade by transient transfection of vector and helper virus. Some workershave even replaced the HIV Env protein with that of vesicular stomatisvirus (VSV). Insertion of the Env protein of VSV facilitates vectorconcentration as HIV/VSV-G vectors with titres of 5×10⁵ (108 afterconcentration) were generated by transient transfection (Naldini et al1996 Science 272: 263-267). Thus, transient transfection of HIV vectorsmay provide a useful strategy for the generation of high titre vectors(Yee et al 1994 PNAS. 91: 9564-9568).

If the retroviral component includes an env nucleotide sequence, thenall or part of that sequence can be optionally replaced with all or partof another env nucleotide sequence. Replacement of the env gene with aheterologous env gene is an example of a technique or strategy calledpseudotyping. Pseudotyping is not a new phenomenon and examples may befound in WO-A-98/05759, WO-A-98/05754, WO-A-97/17457, WO-A-96/09400,WO-A-91/00047 and Mebatsion et al 1997 Cell 90, 841-847.

Pseudotyping can confer one or more advantages. For example, with thelentiviral vectors, the env gene product of the HIV based vectors wouldrestrict these vectors to infecting only cells that express a proteincalled CD4. But if the env gene in these vectors has been substitutedwith env sequences from other RNA viruses, then they may have a broaderinfectious spectrum (Verma and Somia 1997 Nature 389:239-242). By way ofexample—workers have pseudotyped an HIV based vector with theglycoprotein from VSV (Verma and Somia 1997 ibid). Alternatively, envcan be modified so as to affect (such as to alter) its specificity.

Thus, the term “recombinant retroviral vector” describes an entity (suchas a DNA molecule) which contains sufficient retroviral sequences toallow an RNA transcript of the vector to be packaged in the presence ofessential retroviral proteins into a retroviral particle capable ofinfecting a target cell. Infection of the target cell includes reversetranscription and integration into the target cell genome.

The term “recombinant retroviral vector” also covers a retroviralparticle containing an RNA genome encoded by the DNA molecule. Theretroviral vector will also contain non-viral genes which are to bedelivered by the vector to the target cell. A recombinant retroviralvector is incapable of independent replication to produce infectiousretroviral particles. Usually, a recombinant retroviral vector lacksfunctional gag-pol and/or env genes, or other genes encoding proteinsessential for replication.

The term “targeted retroviral vector” means a recombinant retroviralvector whose ability to infect a cell or to be expressed in the targetcell is restricted to certain cell types within the host organism. Anexample of targeted retroviral vectors is one with a geneticallymodified envelope protein which binds to cell surface molecules foundonly on a limited number of cell types in the host organism. Anotherexample of a targeted retroviral vector is one which contains promoterand/or enhancer elements which permit expression of one or moreretroviral transcripts in only a proportion of the cell types of thehost organism. NS/NOI

The vector system of the present invention comprises a nucleotidesequence (“NS”) coding for an antibody. The system may also comprise anucleotide of Interest (“NOI”) which may optionally encode a protein ofinterest (“POI”)

On occasions in the following text, the NS and NOI may be individuallyor collectively referred to as being a gene.

The NS and NOI can be any suitable nucleotide sequence. For example, theNOI can be, for example, a synthetic DNA or RNA sequence, a natural DNAor RNA sequence, a recombinant DNA or RNA sequence (i.e. prepared by useof recombinant DNA techniques), a cDNA sequence or a partial genomic DNAsequence, including combinations thereof. The NOI may be a sensesequence or an antisense sequence.

There may be a plurality of NSs or NOIs, which may be directly orindirectly joined to each other, or combinations thereof. Thus, theexpressed product may have two or more effector domains (which may bethe same or different) and/or two or more “antibody” domains (which maybe the same or different).

The NS encodes an antibody (see below).

The NOI may encode a protein of interest (“POI”). In this way, thevector system could be used to examine the effect of expression of aforeign gene on the target cells (such as a tumour cell). By way ofexample, the vector system could be used to screen a cDNA library for aparticular effect on specific tumour cells. Alternatively the POI mayhave therapeutic, diagnostic, selection, and/or marker properties (seebelow).

The POIs may be proteins which are secreted from the cell. Alternativelythe NOI expression products may not be secreted and may be active withinthe cell. For some applications, it is preferred for the NOI expressionproduct to demonstrate a bystander effect or a distant bystander effect;that is the production of the expression product in one cell leading tothe modulation of additional, related cells, either neighbouring ordistant (e.g. metastatic), which possess a common phenotype.

The NOI may be capable of blocking or inhibiting the expression of agene in the target cell. For example, the NOI may be an antisensesequence. The inhibition of gene expression using antisense technologyis well known in the art.

The NOIs or a sequence derived from the NOIs may be capable of “knockingout” the expression of a particular gene in the target cell (forexample, a tumour cell). There are several “knock out” strategies knownin the art. For example, the NOI may be capable of integrating in thegenome of the target cell so as to disrupt expression of the particulargene. The NOI may disrupt expression by, for example, introducing apremature stop codon, by rendering the downstream coding sequence out offrame, or by affecting the capacity of the encoded protein to fold(thereby affecting its function).

Included in the scope of the invention are oligonucleotide sequences,anti-sense RNA and DNA molecules and ribozymes, which function todestabilise the mRNA or inhibit translation of gene of interest. Suchnucleotide sequences may be used in conditions where it would bepreferable to decrease gene of interest's nucleotide and protein levels,such as in breast cancer (BRACA genes), Burkiff's Lymphoma (c-myc),colon cancer (tumour suppressor deleted in colon cancer (DCC)) (Huertaet al., 2001, Dig Dis Sci, 46, 1884-91) and others.

The vector system of the present invention could be used to flood targetcells with untranslatable sense or antisense sequences. Even in theabsence of integration into the DNA, such vectors may continue totranscribe RNA molecules until all copies of the vector are disabled byendogenous nucleases. Such transient expression may last for a month ormore with a non-replicating vector and even longer if appropriatereplication or integration elements are integrated into the NOI (such aslentivirus derived LTR sequences and Adeno Associated Virus IRsequences.

Modifications of gene expression can be obtained by designing anti-sensesequences to the control regions of, for example, tumour specific targetgenes, such as the promoters, enhancers, and introns.

Oligonucleotides derived from the transcription initiation site, e.g.,between −10 and +10 regions of the leader sequence, are preferred.Anti-sense RNA and DNA molecules may also be designed to blocktranslation of mRNA by preventing the transcript from binding toribosomes. Similarly, inhibition can be achieved using Hogeboombase-pairing methodology, also known as “triple helix” base pairing.Triple helix pairing compromises the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules.

Alternatively, the NOI may be capable of enhancing or inducing ectopicexpression of a gene in a target cell. The NOI or a sequence derivedtherefrom may be capable of “knocking in” the expression of a particulargene.

Transfected target cells which express a particular gene, or which lackthe expression of a particular gene have applications in drug discoveryand target validation. The expression system could be used to determinewhich genes have a desirable effect on target tumour cells, such asthose genes or proteins which are able to trigger apoptosis in thecells. Equally, if the inhibition or blocking of expression of aparticular gene is found to have a cytotoxic effect on the target tumourcell, this may open up possible therapeutic strategies which ensure thatexpression of the gene is not lost.

An NOI delivered by the vector system may be used for selection ormarker purposes. For example, the NOI may encode for a selection gene,or a marker gene. Many different selectable markers are known in the artand include, but are not limited to, the bacterial neomycin andhygromycin phosphotransferase genes which confer resistance to G418 andhygromycin respectively; a mutant mouse dihydrofolate reductase genewhich confers resistance to methotrexate; the bacterial gpt gene whichallows cells to grow in medium containing mycophenolic acid, xanthineand aminopterin; the bacterial hisD gene which allows cells to grow inmedium without histidine but containing histidinol; the multidrugresistance gene (mdr) which confers resistance to a variety of drugs;and the bacterial genes which confer resistance to puromycin orphleomycin. All of these markers are dominant selectable and allowchemical selection of most cells expressing these genes.

The NOI delivered by the vector system may be a therapeutic gene—in thesense that the gene itself may be capable of eliciting a therapeuticeffect or it may code for a product that is capable of eliciting atherapeutic effect. In a highly preferred aspect of the presentinvention, the NOI encodes for an anti-tumour agent.

The NOI may be or encode a palliative agent, i.e. a compound which mayprovides relief, but not a cure.

In one preferred embodiment, the NOI is capable of encoding a cytotoxicmolecule. In particular, the NOI(s) may encode molecules which enhancetarget cells to perish or which stimulate re-generation and functionalrecovery in the damaged tissue. In another preferred embodiment, the NOIis capable of encoding an enzyme or enzymes responsible for converting apro-drug into its active metabolite.

In accordance with the present invention, suitable NOIs include thosethat are (or can produce entities) of therapeutic and/or diagnosticapplication such as, but not limited to: cytokines, chemokines,hormones, antibodies, engineered immunoglobulin-like molecules, a singlechain antibody, fusion proteins, enzymes, immune co-stimulatorymolecules, immunomodulatory molecules, anti-sense RNA, a transdominantnegative mutant of a target protein, a toxin, a conditional toxin, anantigen, a tumour suppresser protein and growth factors, membraneproteins, vasoactive proteins and peptides, anti-viral proteins andribozymes, and derivatives thereof (such as with an associated reportergroup).

The term “enzyme” as used herein means a reaction catalysing substancemodulating but not limited to protein or polypeptide or a fragment ofsuch protein or polypeptide.

The NOI may also be or encode an antiapoptotic factor or aneuroprotective molecule. The survival of cells during programmed celldeath depends critically on their ability to access “trophic” molecularsignals derived primarily from interactions with other cells. Forexample, the NOI may encode a pro-apoptotic gene such as p53 or it maybe a gene involved in control of the cell death cascade (such as Bcl-2).

The NOI may be a ribozyme. Ribozymes are enzymatic RNA molecules capableof catalysing the specific cleavage of RNA.

Suitable NOIs for use in the treatment or prevention of ischaemic heartdisease include NOIs encoding plasminogen activators. Suitable NOIs forthe treatment or prevention of rheumatoid arthritis or cerebral malariainclude genes encoding anti-inflammatory proteins, antibodies directedagainst tumour necrosis factor (TNF) alpha, and anti-adhesion molecules(such as antibody molecules or receptors specific for adhesionmolecules).

Examples of hypoxia regulatable therapeutic NOIs can be found inPCT/GB95/00322 (WO-A-9521927).

In addition to the therapeutic gene or genes and the expressionregulatory elements described, the delivery system may containadditional genetic elements for the efficient or regulated expression ofthe gene or genes, including promoters/enhancers, translation initiationsignals, internal ribosome entry sites (IRES), splicing andpolyadenylation signals.

The NOI or NOIs may be under the expression control of an expressionregulatory element, usually a promoter or a promoter and enhancer. Theenhancer and/or promoter may be preferentially active in a hypoxic orischaemic or low glucose environment, such that the NOI ispreferentially expressed in the particular tissues of interest, such asin the environment of a tumour, arthritic joint or other sites ofischaemia. Thus any significant biological effect or deleterious effectof the NOI on the individual being treated may be reduced or eliminated.The enhancer element or other elements conferring regulated expressionmay be present in multiple copies. Likewise, or in addition, theenhancer and/or promoter may be preferentially active in one or morespecific cell types—such as any one or more of macrophages, endothelialcells or combinations thereof.

Further examples include respiratory airway epithelial cells,hepatocytes, muscle cells, cardiac myocytes, synoviocytes, primarymammary epithelial cells and post-mitotically terminally differentiatednon-replicating cells such as macrophages and neurons.

The term “promoter” is used in the normal sense of the art, e.g. an RNApolymerase binding site in the Jacob-Monod theory of gene expression.

The term “enhancer” includes a DNA sequence which binds to other proteincomponents of the transcription initiation complex and thus facilitatesthe initiation of transcription directed by its associated promoter.

The promoter and enhancer of the transcription units encoding thesecondary delivery system are preferably strongly active, or capable ofbeing strongly induced, in the primary target cells under conditions forproduction of the secondary delivery system. The promoter and/orenhancer may be constitutively efficient, or may be tissue or temporallyrestricted in their activity. Examples of temporally restrictedpromoters/enhancers are those which are responsive to ischaemia and/orhypoxia, such as hypoxia response elements or the promoter/enhancer of agrp78 or a grp94 gene. One preferred promoter-enhancer combination is ahuman cytomegalovirus (hCMV) major immediate early (MIE)promoter/enhancer combination.

Preferably the promoters of the present invention are tissue specific.That is, they are capable of driving transcription of a NOI or NOI(s) inone tissue while remaining largely “silent” in other tissue types.

The term “tissue specific” means a promoter which is not restricted inactivity to a single tissue type but which nevertheless showsselectivity in that they may be active in one group of tissues and lessactive or silent in another group.

The level of expression of an NOI or NOIs under the control of aparticular promoter may be modulated by manipulating the promoterregion. For example, different domains within a promoter region maypossess different gene regulatory activities. The roles of thesedifferent regions are typically assessed using vector constructs havingdifferent variants of the promoter with specific regions deleted (thatis, deletion analysis). This approach may be used to identify, forexample, the smallest region capable of conferring tissue specificity.

A number of tissue specific promoters, described above, may beparticularly advantageous in practising the present invention. In mostinstances, these promoters may be isolated as convenient restrictiondigestion fragments suitable for cloning in a selected vector.Alternatively, promoter fragments may be isolated using the polymerasechain reaction. Cloning of the amplified fragments may be facilitated byincorporating restriction sites at the 5′ end of the primers.

Promoters suitable for cardiac-specific expression include the promoterfrom the murine cardiac α-myosin heavy chain (MHC) gene. Suitablevascular endothelium-specific promoters include the Et-1 promoter andvon Willebrand factor promoter.

Prostate specific promoters include the 5′flanking region of the humanglandular kallikrein-1 (hKLK2) gene and the prostate specific antigen(hKLK3).

Examples of promoters/enhancers which are cell specific include amacrophage-specific promoter or enhancer, such as CSF-1promoter-enhancer, or elements from a mannose receptor genepromoter-enhancer (Rouleux et a/1994 Exp Cell Res 214:113-119).Alternatively, promoter or enhancer elements which are preferentiallyactive in neutrophils, or a lymphocyte-specific enhancer such as an IL-2gene enhancer, may be used.

The vector system of the present invention may delivery the NS and/orNOI to a target cell. The target cell may be any host cell capable ofexpressing the antibody in vivo (or ex vivo). The target cell may alsobe capable of expressing the POI, or the NOi may be delivered to anothercell for POI expression.

Where the antibody and/or NOI and/or POI exerts an effect (such as atherapeutic effect) this may be on the target cell. Alternatively theantibody and/or NOI and/or POI may exerts an effect on a different cell.The target cell may act as an in situ factory for production of theantibody and/or NOI and/or POI.

The target cell may be a tumour cell.

The target cell may be a precursor cell such as a haematopoietic(preferably myeloid haematopoietic) cell of the monocyte-macrophagelineage or a precursor of such cells such as a CD34-positive stem cell.

Antibody

The vector system of the present invention comprises a NS encoding anantibody.

As used herein, “antibody” includes a whole immunoglobulin molecule or apart thereof or a bioisostere or a mimetic thereof or a derivativethereof or a combination thereof. Examples of a part thereof include:Fab, F(ab)′₂, and Fv. Examples of a bioisostere include single chain Fv(ScFv) fragments, chimeric antibodies, bifunctional antibodies.

The term “mimetic relates to any chemical which may be a peptide,polypeptide, antibody or other organic chemical which has the samebinding specificity as the antibody.

The term “derivative” as used herein includes chemical modification ofan antibody. Illustrative of such modifications would be replacement ofhydrogen by an alkyl, acyl, or amino group.

A whole immunoglobulin molecule is divided into two regions: binding(Fab) domains that interact with the antigen and effector (Fc) domainsthat signal the initiation of processes such as phagocytosis. Eachantibody molecule consists of two classes of polypeptide chains, light(L) chains and heavy (H) chains. A single antibody has two identicalcopies of the L chain and two of the H chain. The N-terminal domain fromeach chain forms the variable regions, which constitute theantigen-binding sites. The C-terminal domain is called the constantregion. The variable domains of the H (V_(H)) and L (V_(L)) chainsconstitute an Fv unit and can interact closely to form a single chain Fv(ScFv) unit. In most H chains, a hinge region is found. This hingeregion is flexible and allows the Fab binding regions to move freelyrelative to the rest of the molecule. The hinge region is also the placeon the molecule most susceptible to the action of protease which cansplit the antibody into the antigen binding site (Fab) and the effector(Fc) region.

The domain structure of the antibody molecule is favourable to proteinengineering, facilitating the exchange between molecules of functionaldomains carrying antigen-binding activities (Fabs and Fvs) or effectorfunctions (Fc). The structure of the antibody also makes it easy toproduce antibodies with an antigen recognition capacity joined tomolecules such as toxins, lymphocytes or growth factors.

Chimeric antibody technology involves the transplantation of whole mouseantibody variable domains onto human antibody constant domains. Chimericantibodies are less immunogenic than mouse antibodies but they retaintheir antibody specificity and show reduced HAMA responses.

In chimeric antibodies, the variable region remains completely murine.However, the structure of the antibody makes it possible to producevariable regions of comparable specificity which are predominantly humanin origin. The antigen-combining site of an antibody is formed from thesix complementarity-determining regions (CDRs) of the variable portionof the heavy and light chains. Each antibody domain consists of sevenantiparallel β-sheets forming a β-barrel with loops connecting theβ-strands. Among the loops are the CDR regions. It is feasible to morethe CDRs and their associated specificity from one scaffolding β-barrelto another. This is called CDR-grafting. CDR-grafted antibodies appearin early clinical studies not to be as strongly immunogenic as eithermouse or chimaeric antibodies. Moreover, mutations may be made outsidethe CDR in order to increase the binding activity thereof, as inso-called humanised antibodies.

Fab, Fv, and single chain Fv (ScFv) fragments with VH and VL joined by apolypeptide linker exhibit specificities and affinities for antigensimilar to the original monoclonal antibodies. The ScFv fusion proteinscan be produced with a non-antibody molecule attached to either theamino or carboxy terminus. In these molecules, the Fv can be used forspecific targeting of the attached molecule to a cell expressing theappropriate antigen. Bifunctional antibodies can also be created byengineering two different binding specificities into a single antibodychain. Bifunctional Fab, Fv and ScFv antibodies may comprise engineereddomains such as CDR grafted or humanised domains.

A large number of monoclonal antibodies and immunoglobulin-likemolecules are known which bind specifically to antigens present on thesurfaces of particular cell types. Procedures for identifying,characterising, cloning and engineering these molecules are wellestablished, for example using hybridomas derived from mice ortransgenic mice, phage-display libraries or scFv libraries. Genesencoding immunoglobulins or immunoglobulin-like molecules can beexpressed in a variety of heterologous expression systems. Largeglycosylated proteins including immunoglobulins are efficiently secretedand assembled from eukaryotic cells, particularly mammalian cells.Small, non-glycosylated fragments such as Fab, Fv, or scFv fragments canbe produced in functional form in mammalian cells or bacterial cells.

The antibody used in the present invention may be derived from a humanantibody or an engineered, humanised rodent antibody such as aCDR-grafted antibody or may be derived from a phage-display library.

The antigen-binding domain may be comprised of the heavy and lightchains of an immunoglobulin, expressed from separate genes, or may usethe light chain of an immunoglobulin and a truncated heavy chain to forma Fab or a F(ab)′₂ fragment. Alternatively, truncated forms of bothheavy and light chains may be used which assemble to form a Fv fragment.An engineered scFv fragment may also be used, in which case, only asingle gene is required to encode the antigen-binding domain.

As is well known, antibodies play a key role in the immune system. Inbrief, the immune system works in three fundamentally different ways: byhumoral immunity, by cellular immunity and by secretion of stimulatoryproteins, called lymphokines. Humoral immunity relies on proteinscollectively called immunoglobulin which constitute about 20% of theproteins in the blood. A singly immunoglobulin molecule is called anantibody but “antibody” is also used to mean many different moleculesall directed against the same target molecule. Humoral immunity alsoinvolves complement, a set of proteins that are activated to killbacteria both nonspecifically and in conjunction with antibody.

In cellular immunity, intact cells are responsible for recognition andelimination reactions. The body's first line of defense is therecognition and killing of microorganisms by phagocytes, cellsspecialised for the ingestion and digestion of unwanted material. Thesecells include neutrophils and macrophages. A key role of antibodies isto help phagocytes recognise and destroy foreign materials.

In order to perform these functions, the antibody is divided into tworegions: binding (Fab) domains that interact with the antigen andeffector (Fc) domains that signal the initiation of processes such asphagocytosis. Each antibody molecule consists of two classes ofpolypeptide chains, light (L) chains and heavy (H) chains. A singleantibody has two identical copies of the L chain and two of the H chain.The N-terminal domain from each chain forms the variable regions, whichconstitute the antigen-binding sites. The C-terminal domain is calledthe constant region. The variable domains of the H (V_(H)) and L (V_(L))chains constitute an Fv unit and can interact closely to form a singlechain Fv (ScFv) unit. In most H chains, a hinge region is found. Thishinge region is flexible and allows the Fab binding regions to movefreely relative to the rest of the molecule. The hinge region is alsothe place on the molecule most susceptible to the action of proteasewhich can split the antibody into the antigen binding site (Fab) and theeffector (Fc) region.

The domain structure of the antibody molecule is favourable to proteinengineering, facilitating the exchange between molecules of functionaldomains carrying antigen-binding activities (Fabs and Fvs) or effectorfunctions (Fc). The structure of the antibody also makes it easy toproduce antibodies with an antigen recognition capacity joined tomolecules such as toxins, lymphocytes or growth factors.

Monoclonal antibodies are homogenous antibodies of the same antigenicspecificity representing the product of a single clone ofantibody-producing cells. It was recognised that monoclonal antibodiesoffered the basis for human therapeutic products. However, althoughmouse antibodies are similar to human antibodies, they are sufficientlydifferent that they are recognised by the immune system as foreignbodies, thereby giving rise to an immunological response.

This human-anti-mouse-antibody (HAMA) response limits the usefulness ofmouse antibodies as human therapeutic products.

Chimeric antibody technology involves the transplantation of whole mouseantibody variable domains onto human antibody constant domains. Chimericantibodies are less immunogenic than mouse antibodies but they retaintheir antibody specificity and show reduced HAMA responses.

In chimeric antibodies, the variable region remains completely murine.However, the structure of the antibody makes it possible to producevariable regions of comparable specificity which are predominantly humanin origin. The antigen-combining site of an antibody is formed from thesix complementarity-determining regions (CDRS) of the variable portionof the heavy and light chains. Each antibody domain consists of sevenantiparallel β-sheets forming a β-barrel with loops connecting theβ-strands. Among the loops are the CDR regions. It is feasible to morethe CDRs and their associated specificity from one scaffolding β-barrelto another. This is called CDR-grafting. CDR-grafted antibodies appearin early clinical studies not to be as strongly immunogenic as eithermouse or chimaeric antibodies. Moreover, mutations may be made outsidethe CDR in order to increase the binding activity thereof, as inso-called humanised antibodies.

Fab, Fv, and single chain Fv (ScFv) fragments with VH and VL joined by apolypeptide linker exhibit specificities and affinities for antigensimilar to the original monoclonal antibodies. The ScFv fusion proteinscan be produced with a nonantibody molecule attached to either the aminoor carboxy terminus. In these molecules, the Fv can be used for specifictargeting of the attached molecule to a cell expressing the appropriateantigen. Bifunctional antibodies can also be created by engineering twodifferent binding specificities into a single antibody chain.Bifunctional Fab, Fv and ScFv antibodies may comprise engineered domainssuch as CDR grafted or humanised domains.

In virally directed enzyme therapy (VDEPT), a foreign gene is deliveredto normal and cancerous cells by a viral vector—such as a retroviralvector. The foreign gene codes for an enzyme that can convert anon-toxic prodrug (eg 5-fluorocytosine) to a toxic metabolite(5-fluorouracil) that will kill those cells making it (Sikora et al 1994Ann New York Acad Sci 71b: 115-124). If the promoter utilised is tumourspecific, then the toxic product will only be synthesised in the tumourcells. Studies in animal models have demonstrated that this type oftreatment can deliver up to 50-fold more drug than by conventional means(Connors and Knox 1995 Stem Cells 13: 501-511). A variation of thistechnique uses tumour associated antibodies conjugated to prodrugconverting enzymes to provide specific delivery to tumours. This methodis referred to as antibody-directed enzyme prodrug therapy (ADEPT)(Maulik S and Patel S D “Molecular Biotechnology” 1997 Wiley-Liss Inc pp45).

A large number of monoclonal antibodies and immunoglobulin-likemolecules are known which bind specifically to antigens present on thesurfaces of particular cell types such as tumour cells. Procedures foridentifying, characterising, cloning and engineering these molecules arewell established, for example using hybridomas derived from mice ortransgenic mice, phage-display libraries or scFv libraries. Genesencoding immunoglobulins or immunoglobulin-like molecules can beexpressed in a variety of heterologous expression systems. Largeglycosylated proteins including immunoglobulins are efficiently secretedand assembled from eukaryotic cells, particularly mammalian cells.Small, non-glycosylated fragments such as Fab, Fv, or scFv fragments canbe produced in functional form in mammalian cells or bacterial cells.

The immunoglobulin or immunoglobulin-like molecule may be derived from ahuman antibody or an engineered, humanised rodent antibody such as aCDR-grafted antibody or may be derived from a phage-display library ormay be a synthetic immunoglobulin-like molecule.

The antigen-binding domain may be comprised of the heavy and lightchains of an immunoglobulin, expressed from separate genes, or may usethe light chain of an immunoglobulin and a truncated heavy chain to forma Fab or a F(ab)′₂ fragment. Alternatively, truncated forms of bothheavy and light chains may be used which assemble to form a Fv fragment.An engineered scFv fragment may also be used, in which case, only asingle gene is required to encode the antigen-binding domain. In onepreferred aspect, the antigen-binding domain is formed from a Fv or ascFv.

When a pathogen invades the body, lymphocytes respond with three typesof reaction. The lymphocytes of the humoral system (B cells) secreteantibodies that can bind to the pathogen, signalling its degradation bymacrophages and other cells. The lymphocytes of the cellular system (Tcells) carry out two major types of functions. Cytotoxic T lymphocytes(CTLs) develop the ability to directly recognise and kill the cellsinfected by the pathogen. Helper T cells (TH cells) independentlyrecognise the pathogen and secrete protein factors (lymphokines) thatstimulate growth and responsiveness of B cells, T cells, andmacrophages, thus greatly strengthening the power of the immuneresponse.

In a preferred embodiment, the antibody comprises IgG and/or IgE, or apart thereof, or a bioisostere thereof.

In a more preferred embodiment, the antibody comprises IgG, or a partthereof, or a bioisostere thereof.

5T4

Preferably the antibody recognises a trophoblast cell surface antigen.

Preferably the antibody recognises the 5T4 antigen.

The trophoblast cell surface antigen, originally defined by monoclonalantibody 5T4 (Hole and Stern 1988 Br. J. Cancer 57; 239-246), isexpressed at high levels on the cells of a wide variety of humancarcinomas (Myers et al. 1994 J. Biol. Chem. 269; 9319-9324) but, innormal tissues of non-pregnant individuals, is essentially restricted tolow level expression on a few specialised epithelia (Myers et al. ibid.and references therein). The 5T4 antigen has been implicated incontributing to the development of metastatic potential and thereforeantibodies specifically recognising this molecule may have clinicalrelevance in the treatment of tumours expressing the antigen.

The variable region of the 5T4 monoclonal antibody can also be humanisedby a number of techniques, which are known in the art, includinggrafting of the CDR region sequences on to a human backbone. These canthen be used to construct an intact humanised antibody or a humanisedsingle chain antibody (Sab), such as an ScFv coupled to an Fc region(see Antibody Engineering: a practical approach, ed McCafferty et al.1996 OUP).

Here the term Sab is not limited to just a human or a humanised singlechain antibody. Preferably, however the Sab is a human single chainantibody or a humanised single chain antibody, or part thereof—such asScFv coupled to an Fc region.

In accordance with the invention, standard molecular biology techniquesmay be used which are within the level of skill in the art. Suchtechniques are fully described in the literature. See for example;Sambrook et al. (1989) Molecular Cloning; a laboratory manual; Hames andGlover (1985-1997) DNA Cloning: a practical approach, Volumes I—IV(second edition). Methods for the engineering of immunoglobulin genes inparticular are given in McCafferty et al (1996) Antibody engineering: apractical approach.

Diseases

The delivery of one or more one or more therapeutic genes by a deliverysystem according to the present invention may be used alone or incombination with other treatments or components of the treatment.

For example, the delivery system of the present invention may be used todeliver one or more NOI(s) useful in the treatment of the disorderslisted in WO-A-98/05635. For ease of reference, part of that list is nowprovided: cancer, inflammation or inflammatory disease, dermatologicaldisorders, fever, cardiovascular effects, haemorrhage, coagulation andacute phase response, cachexia, anorexia, acute infection, HIVinfection, shock states, graft-versus-host reactions, autoimmunedisease, reperfusion injury, meningitis, migraine and aspirin-dependentanti-thrombosis; tumour growth, invasion and spread, angiogenesis,metastases, malignant, ascites and malignant pleural effusion; cerebralischaemia, ischaemic heart disease, osteoarthritis, rheumatoidarthritis, osteoporosis, asthma, multiple sclerosis, neurodegeneration,Alzheimer's disease, atherosclerosis, stroke, vasculitis, Crohn'sdisease and ulcerative colitis; periodontitis, gingivitis; psoriasis,atopic dermatitis, chronic ulcers, epidermolysis bullosa; cornealulceration, retinopathy and surgical wound healing; rhinitis, allergicconjunctivitis, eczema, anaphylaxis; restenosis, congestive heartfailure, endometriosis, atherosclerosis or endosclerosis.

In addition, or in the alternative, the delivery system of the presentinvention may be used to deliver one or more NOI(s) useful in thetreatment of disorders listed in WO-A-98/07859. For ease of reference,part of that list is now provided: cytokine and cellproliferation/differentiation activity; immunosuppressant orimmunostimulant activity (e.g. for treating immune deficiency, includinginfection with human immune deficiency virus; regulation of lymphocytegrowth; treating cancer and many autoimmune diseases, and to preventtransplant rejection or induce tumour immunity); regulation ofhaematopoiesis, e.g. treatment of myeloid or lymphoid diseases;promoting growth of bone, cartilage, tendon, ligament and nerve tissue,e.g. for healing wounds, treatment of burns, ulcers and periodontaldisease and neurodegeneration; inhibition or activation offollicle-stimulating hormone (modulation of fertility);chemotactic/chemokinetic activity (e.g. for mobilising specific celltypes to sites of injury or infection); haemostatic and thrombolyticactivity (e.g. for treating haemophilia and stroke); antiinflammatoryactivity (for treating e.g. septic shock or Crohn's disease); asantimicrobials; modulators of e.g. metabolism or behaviour; asanalgesics; treating specific deficiency disorders; in treatment of e.g.psoriasis, in human or veterinary medicine.

In addition, or in the alternative, the delivery system of the presentinvention may be used to deliver one or more NOI(s) useful in thetreatment of disorders listed in WO-A-98/09985. For ease of reference,part of that list is now provided: macrophage inhibitory and/or T cellinhibitory activity and thus, anti-inflammatory activity; anti-immuneactivity, i.e. inhibitory effects against a cellular and/or humoralimmune response, including a response not associated with inflammation;inhibit the ability of macrophages and T cells to adhere toextracellular matrix components and fibronectin, as well as up-regulatedfas receptor expression in T cells; inhibit unwanted immune reaction andinflammation including arthritis, including rheumatoid arthritis,inflammation associated with hypersensitivity, allergic reactions,asthma, systemic lupus erythematosus, collagen diseases and otherautoimmune diseases, inflammation associated with atherosclerosis,arteriosclerosis, atherosclerotic heart disease, reperfusion injury,cardiac arrest, myocardial infarction, vascular inflammatory disorders,respiratory distress syndrome or other cardiopulmonary diseases,inflammation associated with peptic ulcer, ulcerative colitis and otherdiseases of the gastrointestinal tract, hepatic fibrosis, livercirrhosis or other hepatic diseases, thyroiditis or other glandulardiseases, glomerulonephritis or other renal and urologic diseases,otitis or other oto-rhino-laryngological diseases, dermatitis or otherdermal diseases, periodontal diseases or other dental diseases, orchitisor epididimo-orchitis, infertility, orchidal trauma or otherimmune-related testicular diseases, placental dysfunction, placentalinsufficiency, habitual abortion, eclampsia, pre-eclampsia and otherimmune and/or inflammatory-related gynaecological diseases, posterioruveitis, intermediate uveitis, anterior uveitis, conjunctivitis,chorioretinitis, uveoretinitis, optic neuritis, intraocularinflammation, e.g. retinitis or cystoid macular oedema, sympatheticophthalmia, scleritis, retinitis pigmentosa, immune and inflammatorycomponents of degenerative fondus disease, inflammatory components ofocular trauma, ocular inflammation caused by infection, proliferativevitreo-retinopathies, acute ischaemic optic neuropathy, excessivescarring, e.g. following glaucoma filtration operation, immune and/orinflammation reaction against ocular implants and other immune andinflammatory-related ophthalmic diseases, inflammation associated withautoimmune diseases or conditions or disorders where, both in thecentral nervous system (CNS) or in any other organ, immune and/orinflammation suppression would be beneficial, Parkinson's disease,complication and/or side effects from treatment of Parkinson's disease,AIDS-related dementia complex HIV-related encephalopathy, Devic'sdisease, Sydenham chorea, Alzheimer's disease and other degenerativediseases, conditions or disorders of the CNS, inflammatory components ofstokes, post-polio syndrome, immune and inflammatory components ofpsychiatric disorders, myelitis, encephalitis, subacute sclerosingpan-encephalitis, encephalomyelitis, acute neuropathy, subacuteneuropathy, chronic neuropathy, Guillaim-Barre syndrome, Sydenham chora,myasthenia gravis, pseudo-tumour cerebri, Down's Syndrome, Huntington'sdisease, amyotrophic lateral sclerosis, inflammatory components of CNScompression or CNS trauma or infections of the CNS, inflammatorycomponents of muscular atrophies and dystrophies, and immune andinflammatory related diseases, conditions or disorders of the centraland peripheral nervous systems, post-traumatic inflammation, septicshock, infectious diseases, inflammatory complications or side effectsof surgery, bone marrow transplantation or other transplantationcomplications and/or side effects, inflammatory and/or immunecomplications and side effects of gene therapy, e.g. due to infectionwith a viral carrier, or inflammation associated with AIDS, to suppressor inhibit a humoral and/or cellular immune response, to treat orameliorate monocyte or leukocyte proliferative diseases, e.g. leukaemia,by reducing the amount of monocytes or lymphocytes, for the preventionand/or treatment of graft rejection in cases of transplantation ofnatural or artificial cells, tissue and organs such as cornea, bonemarrow, organs, lenses, pacemakers, natural or artificial skin tissue.

The subject treated by the method of the present invention may be ahuman or animal subject. Preferably the subject is a mammalian subject,more preferably a human subject.

In one embodiment of the present invention, the disease to be treated isnot cancer.

In another embodiment of the present invention, the disease to betreated is not a virally caused disease.

Tumours

In another embodiment, the disease is a cancer.

Preferably the antibody recognises a tumour. The antibody may be atumour interacting protein, i.e. specific for a tumour. The antibody maybe able to bind specifically to a tumour, and be a tumour bindingprotein (TBP). Preferably the antibody is capable of interactingspecifically with at least one tumour associated cell surface molecule.

The vector system may also comprise an NOI, optionally encoding a POI.In use the vector system may be capable of delivering the NOI and/or thePOI to the interior of a tumour mass.

In addition to cancerous cell, the cell types present within a tumourmass include but are not limited to macrophages, lymphocytes, tumourinfiltrating lymphocyes, endothelial cells etc.

The POI may also comprise at least one tumour binding domain capable ofinteracting with at least one tumour associated cell surface molecule(“TACSM”).

In accordance with the present invention the TACSM can include but isnot limited to a cell surface molecule which plays a role in tumour cellgrowth, migration or metastasis, a receptor for adhesive proteins suchas the integrin vitronectin receptor, a growth factor receptor (such asepidermal growth factor (EGF) receptor, platelet-derived growth factor(PDGF) receptor, fibroblast-derived growth factor (FDGF) receptor, nervegrowth factor receptor, insulin-like growth factor (IGF-1) receptor; aplasminogen activator; a metalloproteinase (such as collagenase) 5T4antigen; a tumour specific carbohydrate moiety; an oncofetal antigen; amucin; a growth factor receptor; a glycoprotein; and an antigenrestricted in its tissue distribution.

Preferably the TACSM is selectively expressed on one cell type or on arestrictive number of cell types.

Preferably in use the vector system is capable of delivering an NOIand/or POI to a selective tumour site.

Examples of a TBP include: an adhesion molecule such as Intercellularadhesion molecule, ICAM-1, ICAM-2, LFA-1, LFA-2, LFA-3, LECAM-1, VLA-4,ELAM, N-CAM, N-cadherin, P-Selectin, CD44 and its variant isoforms (inparticular CD44v6, CD44v7-8), CD56; a growth factor receptor ligand suchepidermal growth factor (EGF), Platelet-derived growth factor (PDGF),Fibroblast-derived growth factor (FDGF), Nerve growth factor,vasopressin, insulin, insulin-like growth factor (IGF-1), hepatocytegrowth factor, nerve growth factor, human growth factor, brain derivedgrowth factor, ciliary neutrophic factor, glial cell line-derived growthfactor; heavy and light chain sequences from an immunoglobulin (Ig)variable region (from human and animal sources), engineered antibody orone from a phage display library. A phage display library is a techniqueof expressing immunoglobulin genes in bacteriophage has been developedas a means for obtaining antibodies with the desired bindingspecificities. Expression systems, based on bacteriophage lambda, andmore recently filamentous phage have been developed. The bacteriophageexpression systems can be designed to allow heavy and light chains toform random combinations which are tested for their ability to bind thedesired antigen.

The TBP may contain an effector domain which is activated on binding ofthe TPB to the TASCM. The effector domain or domains may be activated onbinding of the TBP to a TASCM leading to inhibition of tumour cellproliferation, survival or dissemination. The effector domain maypossess enzymatic activity (such as a pro-drug activating enzyme) or theeffector domain may include a toxin, or an immune enhancer, such as acytokine/lymphokine such as those listed above.

Preferably the TBP comprises one or more binding domains capable ofinteracting with one or more TACSMs which are present on the cancerouscells—which TACSMs may be the same or different.

The term “interacting” includes direct binding, leading to a biologicaleffect as a result of such binding.

Preferably the vector system is used to deliver the antibody and/or anNOI and/or POI ex vivo and/or in vivo to a tumour.

The vector system of the present invention is useful in gene therapy fordelivering the antibody and/or an NOI and/or POI to a selective site.

In a preferred aspect, the present invention relates to the delivery ofTBP-encoding genes to the site of a tumour. This has considerableadvantages for medical applications (such as therapeutic applications)in which TBPs are indicated since it circumvents a number of problemsassociated with delivery of proteins systemically in humans.

In contrast to the problems associated with production and delivery ofproteins, the methods of the invention allow the delivery of genes tothe site of the tumour, thus circumventing a number of productionproblems. The TBPs are thereby produced in situ in the autologous humancells, which serve as a local factory for the production of thegene-based medicament (such as a therapeutic). This has significantadvantages in minimising systemic toxicity. The activity of the proteinis maximal since the glycosylation of the protein shows a human patternappropriate to the individual being treated.

The methods of the invention can be used in conjunction with directinjection into the site of the tumour or systemic delivery of, forexample targeted vectors or engineered haematopoietic (preferablymyeloid haematopoietic) cells or their progenitors. Systemic deliverymay be particularly advantageous in a number of indications,particularly in the treatment of disseminated disease. In these casesthe gene delivery system or engineered cells can be administeredintravenously by bolus injection or by infusion in a suitableformulation. A pharmaceutically acceptable formulation may include anisotonic saline solution, a buffered saline solution or a tissue-culturemedium. Additional formulatory agents may be included such aspreservative or stabilising agents.

Costimulatory Molecules

Lymphocytes require at least two distinct signals in order to respond toantigens by activation of effector functions (Bretscher and Cohn 1970Science 169: 1042-1049; Crabtree 1989 Science 243: 355-361). The primarysignal is specific for antigen. For B-lymphocytes, the B-cell antigenreceptor (surface immunoglobulin) recognises three-dimensional epitopeson a variety of macromolecules. For T-lymphocytes, the T-cell receptor(TCR) recognises peptide antigens displayed on the surface ofantigen-presenting cells by proteins of the major histocompatability(MHC) family (Weiss et al. 1986 Ann. Rev. Immunol. 4: 593-619).

Stimulation of the primary signal in isolation normally leads toapoptosis (programmed cell death) of the lymphocyte or leads to theestablishment of a state of sustained unresponsiveness or anergy (Weisset al. supra). In order to achieve activation of the lymphocyte,accessory signals are required which may be delivered by cytokines or bycell-surface co-stimulatory ligands present on antigen-presenting cells(APC).

There are a number of such co-stimulatory molecules now identifiedincluding adhesion molecules, LFA-3, ICAM-1, ICAM-2. Majorco-stimulatory molecules present on APC are the members of the B7 familyincluding B7-1 (CD80), B7-2 (CD86) and B7-3. These molecules are ligandsof co-stimulatory receptors on lymphocytes including CD28 (WO92/00092),probably the most significant co-stimulatory receptor for restingT-cells. Different members of the B7 family of glycoproteins may deliversubtly different signals to T-cells (Nunes et al. 1996 J. Biol. Chem.271: 1591-1598).

Established tumours, despite the fact that they commonly express unusualantigens on their surfaces, are poorly immunogenic. It has beenpostulated previously that one method for stimulating immune recognitionof tumour cells would be to enhance antigen presentation andco-stimulation of lymphocytes in the context of tumour antigens.Transfection of the genes encoding B7-1 and B7-2, alone or incombination with cytokines, have been shown to enhance the developmentof immunity to experimental tumours in animal models (e.g. Leong et al.1997 Int. J. Cancer 71: 476-482; Zitvogel et al. 1996 Eur. J. Immunol.26:1335-1341; Cayeux et al. 1997 J. Immunol. 158:2834-2841). However, intranslating these results into a practical treatment for human cancer,there are a number of significant problems to be overcome. A majorproblem in such studies is the need to deliver B7 genes in vivo to alarge number of cells of the tumour to achieve efficacy. A secondproblem is that it is important to target expression of B7 to the tumourcells to avoid inappropriate immune cell activation directed againstother cell types.

This aspect of the present invention solves these specific problems bydelivering a gene encoding a secreted co-stimulatory molecule (“SCM”)with binding affinity for a tumour antigen. In this way, a relativelysmall number of transfected cells within the tumour act as a localfactory to produce the co-stimulatory molecule which is shed from theproducer cell and binds to other cells in the tumour. The aspect of thepresent invention has the additional advantage that tumour cells neednot be the target for transfection.

The SCM of the invention is a novel engineered fusion protein comprisinga signal peptide for secretion from mammalian cells, at least oneantigen-binding domain from an immunoglobulin or an immunoglobulin-likemolecule and at least one further domain which acts as a co-stimulatorysignal to a cell of the immune system. The use of combinations of SCMscontaining different co-stimulatory domains is also envisaged. The SCMsare produced by expression of SCM-encoding genes in the autologous cellsof the individual to be treated and hence any post-translationalmodifications added to the protein by the host cell are authentic andprovide fully functional protein and appropriate pharmacokinetics.WO-A-92/00092 describes truncated forms of B7-1, derived by placing atranslation stop codon before the transmembrane domain, secreted frommammalian cells. In that particular case, a heterologous signal peptidefrom the Oncostatin M gene was used. WO-A92/00092 also describes fusionproteins which contain the extracellular domain of B7-1 fused to the Fcregion of an immunoglobulin. Such molecules can bind to CD28 on T-cellsand serve to stimulate T-cell proliferation. However such stimulationoccurs only to a moderate extent unless the B7 or B7-derivative isimmobilised on a solid surface.

Gerstmayer et al. (1997 J. 1 mmol. 158: 4584-4590) describes a fusion ofB7-2 to an scFv specific for ErbB2 followed by a myc epitope tag andpolyhistidine tag which is secreted when expressed in the yeast Pichiapastoris. This molecule retained binding for antigen and co-stimulatedproliferation of T-cells prestimulated with PMA and IL-2. However,glycosylation of such a molecule is of the yeast type, which is likelyto lead to inappropriate pharmacokinetics in humans.

Thus in a preferred embodiment, the NOI encodes a co-stimulatorymolecule or domain thereof. The co-stimulatory molecule or domainthereof may have binding affinity for a tumour antigen. The NS and theNOI may be linked and/or the antibody and POI (which, in thisembodiment, comprises a co-stimulatory molecule or domain thereof) maybe linked.

In accordance with the present invention, any suitable co-stimulalatorydomain(s) may be used. By way of example, co-stimulatory domains can bechosen from extracellular portions of the B7 family of cell-surfaceglycoproteins, including B7-1, B7-2 and B7-3 or other co-stimulatorycell surface glycoproteins such as but not limited to co-stimulatoryreceptor-ligand molecules including CD2/LFA-3, LFA-1/ICAM-1 and ICAM-3.Studies have demonstrated that T cell co-stimulation by monocytes isdependent on each of two receptor ligand pathways CD2/LFA-3 andLFA-1/ICAM-1 (Van Seventer et al 1991 Eur J Immunol 21: 1711-1718). Inaddition, it has been shown that ICAM-3, the third LFA-1counterreceptor, is a co-stimulatory molecule for resting and activatedT lymphocytes (Hernandez-Caselles et al 1993 Eur J Immunol 23:2799-2806).

Other possible co-stimulatory molecules may include a novel glycoproteinreceptor designated SLAM, has been identified which, when engaged,potentiates T-cell expansion in a CD28-independent manner and induces aTh0/Th1 cytokine production profile (Cocks et al 1995 Nature 376:260-263).

CD6, a cell surface glycoprotein, has also been shown to function as aco-stimulatory and adhesion receptor on T cells. Four CD6 isoforms(CD6a, b, c, d) have been described (Kobarg et al 1997 Eur J Immunol 27:2971-2980). A role for the very late antigen (VLA-4) integrin in theactivation of human memory B cells has also been suggested (Silvy et al1997 Eur J Immunol 27: 2757-2764). Endothelial cells also provide uniqueco-stimulatory signals that affect the phenotype of activated CD4+ Tcells (Karmann et al 1996 Eur J Immunol 26: 610-617). A B3 protein,present on the surface of lipopolysaccharide-activated B cells, whichcan provide co-stimulation to resting T cells leading to a predominantrelease of interleukin (IL)-4 and IL-5 and negligible amounts of IL-2and interferon gamma has been described (Vinay et al 1995 J Biol Chem270: 23429-23436). The co-expression of a novel co-stimulatory T cellantigen (A6H) on T cells and tumour cells has suggested a possiblefunction related to common properties of these cells (Labuda et al 1995Int Immunol 7: 1425-1432).

In one preferred embodiment of the invention, the co-stimulatory domainis a portion of B7-1 or B7-2, more preferably the complete extracellularportion of B7-1 or B7-2.

The SCM is formed by expression of a novel gene encoding a fusionprotein containing the antigen-binding domain or domains and theco-stimulatory domain or domains. If the antigen-binding domain iscomprised of a heavy and a light chain, the co-stimulatory domain isfused to one or other of the immunoglobulin chains, preferably to theheavy chain. If the antigen-binding domain is a scFv, the co-stimulatorydomain is fused to the scFv. The domains can be placed in the order(N-terminus to C-terminus): antigen-binding domain followed byco-stimulatory domain; or co-stimulatory domain followed byantigen-binding domain. Preferably, the co-stimulatory domain is placedat the N-terminus followed by the antigen-binding domain. A signalpeptide is included at the N-terminus, and may be for example thenatural signal peptide of the co-stimulatory extracellular domain. Thedifferent domains may be separated by additional sequences, which mayresult from the inclusion of convenient restriction-enzyme cleavagesites in the novel gene to facilitate its construction, or serve as apeptide spacer between the domains, or serve as a flexible peptidelinker or provide another function. Preferably the domains are separatedby a flexible linker.

Two or more different genes encoding different SCMs may be used toachieve improved co-stimulation, or both co-stimulation of naïve T-cellsand induction of memory responses. For example a gene encoding an SCMcontaining the B7-1 extracellular domain may be administered with a geneencoding an SCM containing the B7-2 extracellular domain.

Thus in one aspect of the invention, there is provided one or moregenetic vectors capable of expressing in mammalian cells one or moresecreted co-stimulatory molecules, each secreted co-stimulatory moleculecomprising at least one antigen-binding domain and at least one domainfrom the extracellular portion of a cell-surface co-stimulatorymolecule. The co-stimulatory domain may be obtained from a moleculeexpressed on the surface of an antigen-presenting cell such as a B7family member. Preferably the co-stimulatory domain is from B7-1, B7-2or B7-3. Most preferably it is comprised of B7-1 amino acid residues 1to approximately 215 of the mature B7-1 molecule (described inWO-A-96/00092) or amino acids 1 to approximately 225 of the maturecell-surface form of B7-2 (described in Gerstmeyer et al. 1997 J.Immunol. 158:4584-4590).

The genetic vector according to this aspect of the invention comprisesat least a promoter and enhancer for expression in mammalian cells and apolyadenylation site. Suitable promoters and enhancers include the MIEpromoter-enhancer from human cytomegalovirus or promoters which areexpressed preferentially in cells present within the tumour. Suchpromoter-enhancers include those from the MUC1 gene, the CEA gene or the5T4antigen gene. If two or more SCMs are expressed, the coding regionsfor these may be inserted into two separate vectors or a single vectormay be used to express the two or more genes. In the latter case eachgene is provided with a separate copy of the promoter, or an internalribosome entry site (IRES) is used to separate the two coding sequences.

Effector Domains

The antibody and/or the POI of the present invention may also containone or more effector domains.

The effector domain or domains may be activated on binding of theantibody to a cell surface molecule (“CSM”) leading to inhibition ofcell proliferation, survival or dissemination. The CSM in this aspect ofthe invention is a cell surface molecule for which a specific TBP isavailable such as a tumour specific carbohydrate moiety, an oncofoetalantigen, a mucin, a growth-factor receptor or another glycoprotein. TheCSM is preferably an antigen restricted in its tissue distribution (forexample, it may be restricted to tumour cells). In some instances, theCSM is not shed from the cell surface into the circulation to anappreciable extent. However, shedding may occur. By way of example,shedding of the 5T4 antigen into the stroma can serve to furtherlocalise the NOI and/or the POI to the tumour environment.

The effector domain of the present invention may possess enzymaticactivity and may be for example a pro-drug activating enzyme, or it maybe a non-enzyme domain. Examples of antibodies containing effectordomains with enzyme activity include antibody—enzyme conjugates orfusions. Antibody—enzyme conjugates have been described includingconjugates with alkaline phosphatase (Senter et al., 1988 Proc. Natl.Acad. Sci. 85: 48424846); carboxypeptidase G2 (Bagshawe et al. 1988 Br.J. Cancer 58: 700703); P-lactamase (Shepherd et al 1991 Bioorg. Med.Chem. Lett. 1:21-26); and Penicillin-V-amidase (Kerr et al. 1990 CancerImmunol. Immunother. 31: 202-206. Antibody—enzyme fusions have also beendescribed (Goshorn et al 1993 Cancer Res 53: 2123-2127; Wels et al 1992Bio/Technology 10:1 1 28-1132). Each of these examples can be used inthis aspect of the invention. Additional or alternative enzymes whichmay be included in antibody-enzyme fusions include humanCarboxypeptidase Al or a mutant thereof (Smith et al 1997 J. Biol. Chem.272: 15804-15816); cytosine deaminase (Mullen et al. 1994 Cancer Res.54: 1503-1506); HSV thymidine kinase (Borrelli et al. 1988 Proc. Natl.Acad. Sci. 85: 7572-7576.); nitroreductase; P450-Reductase and a P450.

Preferably the pro-drug activating enzyme domain or domains aregenetically fused to the C-terminus of an immunoglobulin orimmunoglobulin domain such as a scfv or a single-chain antibody orFab-fragment. In a particularly preferred embodiment of this aspect ofthe invention, the immunoglobulin domain or domains are human orhumanised and the enzyme is a human enzymp—such as a Carboxypeptidase aP450 or P450-Reductase. The enzyme may be a mutant enzyme which convertsa pro-drug more efficiently than does the native human enzyme. Inaccordance with the present invention, any enzyme that has utility in anADEPT strategy can be used.

In each case, a suitable pro-drug is used in the treatment of thepatient in combination with the appropriate pro-drug activating enzyme.Examples of pro-drugs include etoposide phosphate (used with alkalinephosphatase Senter et al., 1988 Proc. Nat. Acad. Sci. 85: 4842-4846);5-fluorocytosine (with Cytosine deaminase Mullen et al. 1994 Cancer Res.54: 1503-1506); Doxorubicin-N-p-hydroxyphenoxyacetamide (withPenicillin-V-Amidase (Kerr et al. 1990 Cancer Immunol. Immunother. 31:202-206); Para-N-bis(2-chloroethyl) aminobenzoyl glutamate (withCarboxypeptidase G2); Cephalosporin nitrogen mustard carbamates (withβ-lactamase); SR4233 (with P450 Reducase); Ganciclovir (with HSVthymidine kinase, Borrelli et al. 1988 Proc. Natl. Acad. Sci. 85:7572-7576) mustard pro-drugs with nitroreductase (Friedlos et al. 1997 JMed Chem 40: 1270-1275) and Cyclophosphamide (with P450 Chen et al. 1996Cancer Res 56: 1331-1340).

Alternatively the effector domain may be a non-enzyme domain. Examplesof non-enzyme effector domains include toxins such an exotoxin from apseudomonad bacterium, all or part of a cytokine such as IL-2 or IFNγ,or effector domains from immunoglobulin heavy chains.

In a preferred embodiment of this aspect of the invention, the antibodyor POI contains an effector domain capable of activating macrophage FcgRI, II or III receptors. On binding of the TBP to antigen on the tumourcells, macrophages present within the hypoxic regions of the tumour areactivated to destroy the tumour cells directly by phagocytosis or ADCCor are activated to secrete pro-inflammatory cytokines which serve toenhance the natural immunological response to the tumour. The antibodyor POI may contain an Fc region from an immunoglobulin, a mutant Fcregion, a receptor-binding fragment of the Fc region or may containanother FcR—binding domain.

Preferably the antibody or POI contains an entity, preferably aneffector domain entity, that confers protein stability ex vivo and/or invivo.

In accordance with the present invention, the antibody or POI may be orinclude an intact Fc region from an IgG, (such as human IgG1 or IgG3, ora part thereof.

In one preferred embodiment of this aspect of the invention, theantibody or POI is a Sab (single chain antibody), containing a humanIgG1 constant region and a binding domain which recognises the 5T4antigen.

In a particularly preferred embodiment of this aspect of the invention,the antibody or POI is a Sab (single chain antibody) containing a humanIgG constant region and a binding domain which recognises the 5T4antigen.

The effector domain may be encoded by a portion of a cDNA fused in-frameto the DNA encoding the antibody or POI. Alternatively a genomicfragment containing introns may be used such as a human IgG1 heavy chainconstant region genomic fragment.

Here the term “intron” is used in its normal sense—e.g. an interveningsequence of DNA within a gene which is removed by RNA splicing and so isnot present in the mature messenger RNA and does not code for protein.Introns can be conditional or alternatively spliced in different celltypes.

Introduction of antibody and/or NOI genes into monocytes or macrophagesmay be combined with further treatments to elicit macrophagedifferentiation and activation. For example, cells maintained ex vivomay be treated with cytokines such as IFNγ, CSF-1 or GM-CSF prior tore-introduction into the patient. Alternatively, genes encoding thesecytokines may be introduced into the monocytes/macrophages in the sameor a different vector system in vivo or ex vivo. Consequently in a stillfurther aspect of the invention there is provided a method of treating adisease in a mammal which comprises administering to an individual acombination of a cytokine or a cytokine-encoding gene and one or moreantibody genes.

Additional Functional Component

The antibody and/or the NOI may further comprise at least one additionalfunctional component.

Preferably the additional functional component is selected from any oneor more of a signalling entity (such as a signal peptide), an immuneenhancer, a toxin, or a biologically active enzyme.

In a preferred aspect the POI is a secretable POI. Thus, in this aspectof the present invention, preferably, the additional functionalcomponent is at least an entity capable of causing the POI to besecreted—such as a signalling entity.

The NS and/or NOI may also comprise an additional functional component,such as a promoter.

The term “promoter” is used in the normal sense of the art, e.g. an RNApolymerase binding site in the Jacob-Monod theory of gene expression.

Preferably the vector system comprises a tumour specific promoterenhancer.

Other preferred additional components include entities enablingefficient expression of the antibody and/or POI. For example, theadditional component may be an enhancer. Here, the term enhancerincludes a DNA sequence which binds to other protein components of thetranscription initiation complex and thus facilitates the initiation oftranscription directed by its associated promoter.

As well as promoter(s) and/or enhancer(s) the NS or NOI may comprisetranslation initiation signals, internal ribosome entry sites (IRES),splicing and polyadenylation signals.

The promoter and/or enhancer may be tissue-restricted in its activity.For example a tumour-specific promoter-enhancer, such as a 5T4 antigengene promoter-enhancer or the CEA-gene promoter-enhancer may be used.Alternatively, or additionally, an element or elements for regulatedexpression may be present, such as a hypoxia regulated enhancer. Anexample of a hypoxia regulated expression element (HRE) is a bindingelement for the transcription factor HIF1. The enhancer elements orelements conferring regulated expression may be present in multiplecopies. Preferably, expression of the or a gene (such as a therapeuticgene) is inducible by hypoxia (or low oxygen supply) such as may befound in a tumour mass. Most preferably, the promoter and/or enhancerdirecting expression of the gene (such as a therapeutic gene) containsboth hypoxia-responsive elements and elements which give higherexpression in tumour cells than in neighbouring non-tumour cells.

Pharmaceutical Compositions

The present invention also encompasses a pharmaceutical composition fortreating one or more individuals by gene therapy, wherein thecomposition comprises a therapeutically effective amount of the vectorsystem according to the present invention. The pharmaceuticalcomposition may be for human or animal usage. Typically, a physicianwill determine the actual dosage which will be most suitable for anindividual subject and it will vary with the age, weight and response ofthe particular patient.

The composition may optionally comprise a pharmaceutically acceptablecarrier, diluent, excipient or adjuvant. The choice of pharmaceuticalcarrier, excipient or diluent can be selected with regard to theintended route of administration and standard pharmaceutical practice.The pharmaceutical compositions may comprise as—or in addition to—thecarrier, excipient or diluent any suitable binder(s), lubricant(s),suspending agent(s), coating agent(s), solubilising agent(s), and othercarrier agents that may aid or increase the viral entry into the targetsite (such as for example a lipid delivery system).

Where appropriate, the pharmaceutical compositions can be administeredby any one or more of: inhalation, in the form of a suppository orpessary, topically in the form of a lotion, solution, cream, ointment ordusting powder, by use of a skin patch, orally in the form of tabletscontaining excipients such as starch or lactose, or in capsules orovules either alone or in admixture with excipients, or in the form ofelixirs, solutions or suspensions containing flavouring or colouringagents, or they can be injected parenterally, for exampleintracavemosally, intravenously, intramuscularly or subcutaneously. Forparenteral administration, the compositions may be best used in the formof a sterile aqueous solution which may contain other substances, forexample enough salts or monosaccharides to make the solution isotonicwith blood. For buccal or sublingual administration the compositions maybe administered in the form of tablets or lozenges which can beformulated in a conventional manner.

The invention will now be further described by way of examples, whichare meant to serve to assist one of ordinary skill in the art incarrying out the invention and are not intended in any way to limit thescope of the invention. Reference is made to the following Figures:

FIG. 1 a—which shows a DNA sequence encoding a 5T4 scFv, designated5T4scFv. 1. The sequence of the mature secreted protein is given.

FIGS. 1 b-1 d which show the cDNA sequence encoding 5T4Sab1. Thesequence begins with a HindIII restriction site followed by atranslation initiation signal and a signal peptide.

FIGS. 2 a-2 b—which show the sequence of B7-1.5T4.1

FIG. 3—which shows a diagrammatic representation of two SCMs based onthe B7-1 co-stimulatory domain; FIG. 3 a shows the SCM B7-1.5T4.1 andFIG. 3 b shows B7-1.5T4.2 in which the order of the co-stimulatory andtumour-binding domains are reversed. Sp=signal peptide; B7ec=extracellular domain of B7-1; VI=light chain variable domain of 5T4;Vh=heavy chain variable domain of 5T4.

FIG. 4—which shows the sequence of the extracellular domain of humanB7-2, including the signal peptide sequence. The mature protein beginsat amino acid 17. The B7-2 derived sequence is followed by a flexiblelinker gly-gly-gly-gly-ser.

FIG. 5—which shows pKLink—the (Gly₄Ser)₃ linker in pBluescript II SK(pBS II).

The flexible linker is synthesised as two complementaryoligonucleotides, that are annealed to give restriction enzyme overhangsand then cloned as a double stranded oligonucleotide into pBSII. Theamino acid translation of (glycine₄ serine)₃ is shown in single lettercode below the DNA sequence.

FIG. 6—which shows anti-TNF alpha scFv in pBSII and subsequent additionof the leader sequence

FIG. 7—which shows Leader-IL-5 scFv in pONY 8.1 SM

FIG. 8—which shows Leader-HIV gp120 scFv in pONY 8.18M

FIG. 9—which shows Leader-anti-TNF alpha scFv in pONY 8.1 SM

FIG. 10—which shows Leader-VEGF scFv in pONY 8.1 SM

FIG. 11: Immunostain of CT26-h5T4 tumours injected with Adlac z

FIG. 12: Immunostain of CT26-h5T4 tumours injected with AdB7-scFv

EXAMPLES Example 1 Construction of 5T4 Sab and Retroviral—VectorDelivery to Tumour

The cDNA encoding the murine 5T4 monoclonal antibody is cloned andsequenced by standard techniques (Antibody engineering: a practicalapproach ed McCafferty et al. 1996 OUP). The sequence of the variableregion of the antibody can be used to construct a variety ofimmunoglobulin-like molecules including scFvs. The coding sequence of a5T4 scFv, 5T4scFv.1, is shown in FIG. 1 a. In this molecule, the DNAsequence encodes the Vh from the mouse 5T4 monoclonal antibody followedby a 15 amino acid flexible linker and the VI region of the mouse 5T4antibody. The flexible linker encodes 3 copies of the amino-acidsequence gly-gly-gly-gly-ser and the DNA sequence similarity between therepeats has been minimised to avoid the risk of recombination betweenthe repeats when plasmids containing them are grown in E. coli.

The DNA sequence shown in FIG. 1 a can also be used to construct avariety of single-chain antibodies (Sabs) by coupling scFv-encodingsequences to a sequence encoding a Fc region to form an in-frame fusion.A Sab is constructed using a series of DNA cassettes which can beindependently varied to suit particular purposes.

Cassette 1—Translation initiation signal and signal peptide

In order to achieve correct translation initiation and secretion frommammalian cells, the following sequence is used:

aagcttCCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACA GCTACAGGTGTCCACTCC

This contains a convenient HindIII restriction site for cloning intoexpression vectors (lower case), the consensus translation initiationsignal for mammalian cells (ANNATGPU) and the coding sequence for asignal peptide sequence from an immunoglobulin gene.

Cassette 2—scFv

The sequence of the secreted portion of the 5T4scFv.1 is shown in FIG. 1a. This molecule can be represented as Vh—(gly₄-ser)₃ linker—VI.

5T4 scFv2 consists of the 5T4 variable region sequences connected in theorder VI—flexible linker Vh. In this case the linker encodes the 20amino-acid peptide (gly₄-ser)₄. A longer linker improves assembly of thescFv when the V-region segments are in this order. (Pluckthun et al inAntibody Engineering: a practical approach, ed McCafferty et al. 1996OUP).

Cassette 3—Heavy Chain Constant Region

The sequence of a human g1 constant region genomic clone is given inEllison et al. 1982 Nucl. Acids res. 10: 4071-4079. This sequencecontains constant-region introns in addition to the coding sequence.This is fused in-frame to the 3′-end of one of the scFv sequences fromCassette 2. Vectors for convenient assembly of such constructs aredescribed (Walls et al. 1993 Nucl. Acids Res. 21:2921-2929.

A cDNA of a 5T4 Sab, designated 5T4Sab1 is shown in FIG. 1 b, containingcassettes 1, 2 and 3.

For expression of a 5T4-specific scFv or Sab in human cells, the codingsequence is inserted into the vector pClneo (Promega) under the controlof a strong promoter and polyadenylation signal. The translationinitiation signal and immunoglobulin leader (signal peptide) sequencefrom Cassette 1 at the 5′ end of the coding region ensure efficientsecretion of the scFv or Sab from mammalian cells.

For expression of an intact 1 g, two separate translation cassettes areconstructed, one for the heavy chain and one for the light chain. Theseare separated by an internal ribosome—entry site (IRES) from thepicornavirus FMDV (Ramesh et al. 1996 Nucl. Acids Res. 24: 2697-2700.Alternatively, each cDNA is expressed from a separate copy of the hCMVpromoter (Ward and Bebbington 1995 In Monoclonal Antibodies ed Birch andLennox.Wiley-Liss).

For production of retrovirus capable of expressing 5T4 antibody orimmunoglobulin-like molecules with 5T4 specificity, the gene encoding a5T4-based Sab, or a dicistronic message encoding heavy and light chains,is inserted into a retroviral vector in which retroviral genomictranscripts are produced from a strong promoter such as the hCMV-MIEpromoter. A suitable plasmid is pHIT111 (Soneoka et al. 1995 Nucl. AcidsRes.23; 628-633) and the required gene is inserted in place of the LacZgene using standard techniques. The resulting plasmid, pHIT-5T4.1 isthen transfected into the FLYRD18 or FLYA13 packaging cell lines (Cossetet al. 1995 J. Virol. 69; 7430-7436) and transfectants selected forresistance to G418 at 1 mg/ml. G418-resistant packaging cells producehigh titres of recombinant retrovirus capable of infecting human cells.The virus preparation is then used to infect human cancer cells and canbe injected into tumours in vivo. The 5T4 Sab is then expressed andsecreted from the tumour cells.

In pHIT1111, the MoMLV LTR promoter-enhancer is used for expression ofthe therapeutic gene in the target cell. The vector can also be modifiedso that the therapeutic gene is transcribed from an internalpromoter-enhancer such as one which is active predominantly in thetumour cells or one which contains a hypoxia regulated element. Asuitable promoter is a truncated HSV TK promoter with 3 copies of themouse PGK HRE (Firth et al. 1994 Proc. Natl. Acad. Sci. 91: 6496-6500).

Example 2 Transfection of Macrophages/Monocytes with an ExpressionVector Encoding TBP

Peripheral blood mononuclear cells are isolated from human peripheralblood at laboratory scale by standard techniques procedures (Sandlie andMichaelsen 1996 In Antibody engineering: a practical approach. EdMcCafferty et al. Chapter 9) and at large scale by elutriation (egCeprate from CellPro). Adherent cells (essentially monocytes) areenriched by adherence to plastic overnight and cells can be allowed todifferentiate along the macrophage differentiation pathway by culturingadherent cells for 1-3 weeks.

Monocytes and macrophages are transfected with an expression vectorcapable of expressing TBP in human cells. For constitutive high levelexpression, the TBP is expressed in a vector which utilises the hCMV-MIEpromoter-enhancer, pCl (Promega). For hypoxia-induced expression, thehCMV promoter is replaced by a promoter containing at least one HRE. Asuitable promoter is a truncated HSV TK promoter with 3 copies of themouse PGK HRE (Firth et al. 1994 Proc. Natl. Acad. Sci. 91: 6496-6500).

A variety of transfection methods can be used to introduce vectors intomonocytes and macrophages, including particle-mediated DNA delivery(biolistics), electroporation, cationic agent-mediated transfection (egusing Superfect, Qiagen). Each of these methods is carried out accordingto the manufacturer's instructions, taking into account the parametersto be varied to achieve optimal results as specified by the individualmanufacturer. Alternatively, viral vectors may be used such as defectiveAdenovirus vectors (Microbix Inc or Quantum Biotechnologies Inc).

Example 3 Assay for ADCC Mediated by Macrophages

Cells from primary human tumours or tumour cell lines which have beentransduced with retrovirus expressing TBP are mixed with autologous orheterologous human macrophages, prepared as described in Example 2, foranalysis of ADCC activity mediated by the TBP. Alternatively,macrophages engineered to produce TBP as described in Example 2 can beused to direct ADCC on non-transduced tumour cells.

The assay is carried out according to standard procedures (Sandlie andMichaelsen 1996 In Antibody engineering: a practical approach. EdMcCafferty et al. Chapter 9) with appropriate modifications. Briefly,the effector cells (macrophages or freshly isolated monocytes) aresuspended at 3×10⁶ cells/ml in the appropriate tissue culture medium(DMEM/Hepes, obtained from Life Technologies, containing 1% Foetal CalfSerum). 3×10⁵ tumour target cells, labelled with ⁵¹Cr are placed in eachwell of a round-bottomed microtitre plate in 0.1 ml culture medium.(Note the culture medium can include spent medium from the cellsproducing the TBP). 50 ml effector cells are added to the wells, theplate is centrifuged at 300 g for 2 min and incubated at 37° C. forvarying periods (eg 4 h) in a tissue culture incubator. The supernatantis then harvested by centrifugation and counted in a gamma counter.Results are expressed as percent lysis relative to total chromiumrelease from an equivalent sample of target cells lysed with 0.1%Tween-20. The effector: target cell ratio can be varied in the assay toproduce a titration curve.

For the prior stimulation of macrophage differentiation or priming,cytokines are added to the cultures. IFNg (Sigma) is added at between100 and 5000 U/ml. CSF-1 or GM-CSF (Santa Cruz Biotechnology) can alsobe added at appropriate concentrations.

Example 4 Analysis of Efficacy in Animal Models

Human tumour-derived cell lines and tissues are cultured in vivo ingenetically immunodeficient, “nude” mice according to well establishedtechniques (see for example Strobel et al. 1997 Cancer Res. 57:1228-1232; McLeod et al. 1997 Pancreas 14: 237-248). Syngeneic mousemodels, in which a syngeneic tumour line is introduced into animmunocompetent mouse strain may also be used. These serve as suitableanimal models for evaluating gene delivery systems of the invention.Vectors or engineered cells are administered systemically or directlyinto the tumour and tumour growth is monitored in treated and untreatedanimals. This system is used to define the effective dose range of thetreatments of the invention and the most appropriate route ofadministration.

Example 5 Construction of B7-scFv Fusion Proteins

The extracellular domain of B7-1 is defined by amino-acid residues 1-215of the native human B7-1 protein. This sequence, together with itssignal peptide-encoding sequence, is used to construct secreted fusionproteins which also contain the scFv derived from the 5T4 monoclonalantibody. The sequence of the 5T4 scFv is given in FIG. 1 a.

A DNA coding sequence is constructed using standard molecular biologytechniques which encodes a fusion protein in which the N-terminus of the5T4 scFv is fused after amino acid 215 of human B7-$. The sequence ofthis coding sequence, B7-1.5T4.1, is shown in FIG. 2. The fusion proteincontains a flexible (gly-gly-gly-gly-ser) spacer between the B7-1 and5T4 scFv sequences.

The introduction of a convenient BamH1 restriction site at the end ofthe linker insertion (beginning at nucleotide 733) also allows forfurther linkers to be screened for optimal expression of bifunctionalfusion protein. FIG. 3 indicates the fusion protein in diagrammaticform. It is similarly possible to construct B7-1.5T4.2 (FIG. 3 b) inwhich the scFv is N-terminal and the B7 extracellular domain isC-terminal. In this case only the coding sequence of the mature B7-1(without signal peptide) is required. A signal peptide such as animmunoglobulin leader sequence is added to the N-terminus of the scFv inthis instance.

For fusion proteins which use the co-stimulatory extracellular domain ofB7-2, the signal peptide and extracellular domain of B7-2 is used inplace of B7-1 sequences. FIG. 4 shows the coding sequence of the SCMB7-2.5T4.1co-stimulatory domain. It encodes the first 225 amino acids ofhuman B7-2, preceded by its signal peptide, and a flexible linker(gly4-ser). The BamHI site at the end of this sequence can be used toinsert the domain upstream of the 5T4scFv.1 (see FIG. 3). The sequenceincludes the B7-2 signal peptide which can serve to allow secretion ofthis fusion protein in which the B7-2 domain is at the N-terminus of thefusion protein.

Each engineered cDNA is inserted into the mammalian expression vectorpCl to allow expression in mammalian tissue culture cells. For thispurpose, a linker sequence is added to the 5′-end of the coding sequencewhich introduces a convenient restriction site for insertion into thepolylinker of pCl and adds the translation initiation signal CCACCimmediately adjacent to the first ATG codon. Constructs in pCl aretransfected into a suitable mammalian host cell line such as COS-1 toconfirm secretion of the SCM. The transcription cassette from pCl or anappropriate segment of the transcription cassette is subsequentlysub-cloned into the expression vector to be used as the gene deliverysystem for therapeutic use.

Example 6 Transfection of Macrophages/Monocytes with an ExpressionVector Encoding an SCM

Peripheral blood mononuclear cells are isolated from human peripheralblood at laboratory scale by standard techniques procedures (Sandlie andMichaelsen 1996 In Antibody engineering: a practical approach. EdMcCafferty et al. Chapter 9) and at large scale by elutriation (egCeprate from CelIPro). Adherent cells (essentially monocytes) areenriched by adherence to plastic overnight and cells can be allowed todifferentiate along the macrophage differentiation pathway by culturingadherent cells for 1-3 weeks.

Monocytes and macrophages are transfected with an expression vectorcapable of expressing SCM in human cells. For constitutive high levelexpression, the SCM is expressed in a vector which utilises the hCMV-MIEpromoter-enhancer, pCl (Promega). For hypoxia-induced expression, thehCMV promoter is replaced by a promoter containing at least one HRE. Asuitable promoter is a truncated HSV TK promoter with 3 copies of themouse PGK HRE (Firth et al. 1994 Proc. Natl. Acad. Sci. 91: 6496-6500).

A variety of transfection methods can be used to introduce vectors intomonocytes and macrophages, including particle-mediated DNA delivery(biolistics), electroporation, cationic agent-mediated transfection (egusing Superfect, Qiagen). Each of these methods is carried out accordingto the manufacturer's instructions, taking into account the parametersto be varied to achieve optimal results as specified by the individualmanufacturer. Alternatively, viral vectors may be used such as defectiveAdenovirus vectors (Microbix Inc or Quantum Biotechnologies Inc).

Example 7 Analysis of SCM Binding to CTLA4 and 5T4-antigen ExpressingCells

The B7-1 or B7-2 domains are expected to bind specifically to CD28 andCTLA-4 present on human T-cells. Binding to T-cells or Chinese hamsterovary cells transfected with human CTLA-4 or CD28 is determined usingFACS analysis as follows. 5×10⁵ CTLA-4 expressing target cells orequivalent cells lacking CTLA-4 (untransfected CHO cells) are incubatedwith 0.1 ml culture supernatant from COS-1 cells transiently transfectedwith SCM genes for 1 h at 4° C. The cells are washed and incubate with 1mg monoclonal antibody specific for the B7 domain (eg Mab 9E10) followedby FITC-labelled goat anti-mouse IgG (Pharmingen) and analysis by FACS.

Binding of scFv to 5T4-antigen is similarly assessed using target cellsexpressing 5T4-antigen (5T4-transfected A9 cells) or control cells (A9).

Example 8 Analysis of Co-Stimulatory Activity

An established mouse cell line of Balb/c origin such as HCl 1 cells istransfected with the cDNA encoding human 5T4-antigen (Myers et al. 1994J. Biol. Chem. 269; 9319-9324) inserted in the expression vector pClneo.

Splenic T-cells from Balb/c mice are isolated by standard procedures(Johnstone and Thorpe 1996 In Immunochemistry in Practice. Blackwell.Chapter 4). T-cells are pre-stimulated by incubation for 1-2 days inmedium containing 10 ng/ml PMA (Sigma) and 100 U/ml human IL-2(Boehringer Mannheim). HC11-5T4 cells are incubated at 10⁴ cells/well ofa 96-well tissue culture tray for 2 h with up to 0.1 ml supernatant fromCOS cells transfected with SCM gene. Up to 10⁵ pre-stimulated T-cellsare added to each well, the cells are pulsed with 0.25 mCi/well³H-thymidine and incorporation of ³H-thymidine is measured using aliquid scintillation counter after 24 h.

Incorporation of ³H-thymidine is anticipated to be enhanced by thepresence of SCM.

Example 9 Analysis of Co-Stimulation in Animal Models

HC11 cells transfected with the human 5T4-antigen gene (Example 4) aregrown as tumours in Balb/c mice. SCM genes B7-1.5T4.1 or B7-2.5T4.1 or acombination of both genes, are introduced into the tumour cells prior toimplantation and the growth of the tumours and the growth of controltumours which do not express SCM genes in vivo are monitored.

It is believed that the expression of SCM genes lead to significantreduction in tumour growth.

Example 10 Construction of a B7-1/ScFv, Specific for Human 5T4, FusionProtein

Standard molecular biology techniques are used to construct a fusionprotein consisting of the leader sequence and extracellular domain ofB7-1, fused via a flexible linker to the V_(H) and V_(L) of the murineMab 5T4 specific to human 5T4.

The flexible linker, used to join the extracellular domain of B7.1 andthe ScFv, was constructed by annealing two homologous oligonucleotideswith engineered 5′ Sma I and 3′ Spe I sites—using oligonucleotides upper

5′ GGG GGT GGT GGG AGC GGT GGT GGC GGC AGT GGC GGC GGC GGA A 3′ andlower 5′ CTA GTT CCG CCG CCG CCA CTG CCG CCA CCA CCG CTC CCA CCA CCC CC3′

The linker is cloned into pBluescript (Stratagene) via Sma I and Spe Ito produce pLINK. The signal peptide (sp) and extracellular domain ofmurine B7.1 were ampified by PCR from pLK444-mB7.1 (supplied by R.Germain NIH, USA) via primers that introduce 5′ EcoRI and 3′ Sma Isites—primers forward

5′ C TCG AAT TCC ACC ATG GCT TGC AAT TGT CAG TTG ATG C 3′ reverse 5′ CTCCCC GGG CTT GCT ATC AGG AGG GTC TTC 3′

The B7.1 PCR product was cloned into pLINK via Eco RI and Sma I to formpBS/B7Link.

The V_(H) and V_(L) of the 5T4 specific ScFv was amplified via primers—

forward primer 5′ CTC ACT AGT GAG GTC CAG CTT CAG GAG TC 3′ reverseprimer 5′ CTC GCG GCC GCT TAC CGT TTG ATT TCC AGC TTG GTG CCT CCA CC 3′that introduce 5′ Spe 1 and 3′ Not I sites from pHEN1-5T4 ScFv.PBS/B7Link was digested with Spe I and Not I and ligated with the ScFvto form OBM 233 consisting of the sequence shown as SEQ ID No. 5: B7Link scFv sequence

This fusion can be used to construct a recombinant vector e.g.retrovirus, Lentivirus, adenovirus, poxvirus, vaccinia virus,baculovirus. Such vectors can be used to inject patient tumoursdirectly. To deliver the fusion protein to tumour cells the recombinantvector is used to transduce macrophages/monocytes/CD34+ cells ex vivobefore injection back into patients. These cells will traffic totumours. The ScFv will bind to a specific tumour antigen expressed onthe surface of tumour cells e.g. 5T4 (Myers et al 1994 JBC). B7 is foundon the surface of professional antigen presenting cells e.g.macrophages, dendritic cells and B cells. It interacts with it ligandsCD28 and CTL-A4 located on CD4 and CD8 cells. The simultaneousinteraction of B7-CD28/CTL-A4 and MHC-peptide/T cell receptor leads to apronounced increase in IL-2 which promotes CD8 (cytotoxic T cell)expansion (Linsley P S, Brady W, Grosmaire L, Aruffo A, Damle N K,Ledbetter J A J Exp Med 1991 Mar. 1; 173(3):721-730 Binding of the Bcell activation antigen B7 to CD28 costimulates T cell proliferation and11-2 mRNA accumulation.) Tumour cells that have been B7 tranfected withB7 have been shown retardation in animal models (Townsend S E, Allison JP Science 1993 15; 259(5093):368-370).

Example 11 Transient Expression and Purification of B7-11/scFv and LScFv

For transient expression of B7-1/ScFv the human CMV expression plasmidpClneo (Promega) was used. B7/ScFv was excised from OBM 233 by digestionwith EcoR I/Not I and cloned into pClneo that was previously digestedwith EcoRI/Not I. Transient expression of recombinant protein is made bytransfection of 293T cells with the relevant plasmid using calciumphosphate (Profectin, Promega). Conditions used were similar to thoserecommended by the manufacturer. To reduce bovine serum contaminationserum free optimum media (Gibco BRL). After 36-48 hours transfectionsupernatants were harvested and spun through a Centriprep (Amicon, Glos.UK) 10 filter (all proteins larger than 10 kDa arepurified/concentrated) and a Centricon (Amicon) 10 filter. Supernatantsare concentrated approximately 30 fold.

For B7-1 to be biologically functional it must be able to displaybinding with one of it's natural ligands either CTLA-4 or CD28 found onthe surface of specific populations of T cells (e.g CD4+). Thebiological activity B7-1/ScFv fusion protein was analysed forsimultaneous interaction with its natural ligand CTLA-4 (in the form ofCTLA4-1 g supplied by Ancell, Minn., USA) and A9 cells expressing human5T4. Briefly: approximately 5×10⁵ A9-h5T4 cells were incubated with 100μl of either B7.1/ScFv or LScFv supernatant in a U bottom 96 well plateat 4° C. for 1 hour. After washing cells were incubated with CTLA4-1 g(Ancell) for 1 hour. After washing, bound CTLA4-1 g was detected usingan FITC conjugated anti-mouse Ig (Dako).

Results show obvious binding of CTLA-Ig with the B7-1 extracellulardomain, bound via the ScFv, to the surface of human 5T4 positive A9cells. The lack of binding activity with 5T4 negative A9 cells furtherillustrates that the interaction of B7 with CTLA4-1 g and ScFv with 5T4are specific.

Example 12 ScFv-IgG Fusion Example Construction of ScFv-IgG

The sequence encoding a translation initiation sequence and the humanimmunoglobulin kappa light chain signal peptide is synthesised as twocomplementary single stranded oligonucleotides which when annealed alsocontain an internal Xho/site at the 5′ end and in addition leave a Xba Icompatible 5′ overhang and a Pst I compatible 3′ overhang

ctagactcgagCCACC ATG GGA TGG AGC TGT ATC ATC CTC TTC TTG GTA GCA ACA GCTACA GGT GTC GAC TCC GAG GTC GAG ctgca and g CTG GAG CTC GGA GTG GAC ACCTGT AGC TGT TGC TAC CAA GAAGAG GAT GAT ACA GCT CCA TCC CAT GGTGGctcgagt

This is then cloned into pBluescript II (Stratagene) restricted with XbaI and Pst I to create pBSII/Leader.

The 5T4 scFv is amplified by PCR from pHEN1 using oligonucleotides whichincorporate a Pst I site at the 5′ end of the product and a Hind III atthe 3′ end

GTC CAG CTG CAG CAG TCT GG and CG TTT GAT TTC AAG CTT GGT GC

This is then restricted with those enzymes and inserted intopBSII/Leader restricted with the same enzymes, creatingpBSII/Leader/scFv.

The HIgG 1 constant region is amplified by PCR from the cloned geneusing oligonucleotides which incorporate a Hind III site at the 5′ endand a Xho I site at the 3′ end

gcgc AAG CTT gaa atc aaa cgg GCC TCC ACC AAG GGC CCA and gcgc ctcgag TCATTT ACC CGG AGA CAG GG

This is then restricted with those enzymes and inserted intopBSII/Leader/scFv restricted with the same enzymes, creatingpBSII/Leader/scFv/HG1. The sequence for this construct is shown in theFigures.

This fusion can be used to construct a recombinant vector e.g.retrovirus, Lentivirus, adenovirus, poxvirus, vaccinia virus,baculovirus. Such vectors can be used to inject patient tumoursdirectly. To deliver the fusion protein to tumour cells the recombinantvector is used to transduce macrophages/monocytes/CD34+ cells ex vivobefore injection back into patients. These cells will traffic totumours. The ScFv will bind to a specific tumour antigen expressed onthe surface of tumour cells e.g. 5T4 (Myers et al 1994 JBC). Bound IgGwill promote specific tumour destruction via a collection of mechanismscollectively known as antibody dependent cellular cytotoxicity (Munn etal Can res 1991 ibid, Primus et al 1993 Cancer Res ibid).

Example 13 Construction of ScFv-IgE1 (Human IgE1 Heavy Constant Region)

A similar fusion construct of 5T4 scFv—human IgE constant heavy chain ismade consisting of the sequence shown as SEQ ID No. 6.

This fusion construct is made by amplifying the human IgE1 constantheavy region by PCR cDNA derived from human B-cells RNA by RT andsubsequently using oligonucleotides which incorporate a Hind III site atthe 5′ end and a Xho I site at the 3′ end

gcgc AAG CTT gaa atc aaa cgg GCC TCC ACA CAG AGC CCA and gcgc ctcgag TCATTT ACC GGG ATT TAC AGA

This is then restricted with those enzymes and inserted intopBSII/Leader/scFv restricted with the same enzymes, creatingpBSII/Leader/scFv/HE1.

As described above the ScFv-IgE construct can be incorporated into arecombinant viral vector for use in gene therapy of cancer e.g. injectpatient tissue directly or to transduce patient derivedmacrophages/moncytes/CD34+ cells ex vivo. The fusion protein will besecreted and will bind to tumour cells bearing the antigen that the ScFvis specific for. Binding of IgE to tumour cells should promote a stronghistamine response via activation of mast cells. This will lead to astrong inflammatory response and destruction tumour cells as is reportedfor IgE cytotoxic destruction of parasites e.g. helminth larvae (CapronM 1988 Eosinophils in diseases: receptors and mediators. In progress inallergy and clinical immunology (Proc. 13^(th) Int. Congress of Allergyand Clinical Immunology) Hogrefe & Huber Toronto p6). Such inflammationand tumour destruction should initiate the recruitment of other immuneeffector cells. Past reports indicate that treatment with an MMTVantigen specific IgE Mab leads to protection from a tumour expressingMMTV antigen (Nagy E Istanvan B, Sehon A H 1991 Cancer Immunol.Immunotherapy vol 34:63-69).

Example 14 Construction of B7/EGF B7—EGF Synthetic Gene.

A fusion construct of B7-EGF is made by inserting a PCR productamplified from the region of the gene encoding the mature EGF peptide(see accession number X04571) into pBS/B7 Link. This construct has thesequence shown as SEQ ID No. 7.

Using cDNA derived by RT of RNA isolated from a cell line such the 293human kidney line (ATCC: CRL1573), the DNA is amplified by PCR usingoligonucleotides containing a Spe I restriction enzyme site at theN-terminus and a stop codon and a Not I site at the C-terminus

GG ACT AGT AAT AGT GAG TCT GAA TGT CCC and ATT AGC GGC CGC TTA GCG CAGTTC CCA CCA CTT C

The resulting product is digested with those enzymes and ligated topBS/B7 Link which has been restricted with the same enzymes creatingpBS/B7 Link EGF. The B7 Link EGF cassette is then excised with Eco RIand Not I and inserted into a derivative of pHIT111 (Soneoka et al.,1995, Nucl Acid Res 23; 628) which no longer carries the LacZ gene

An alternative to using ScfV is to use growth factors that have a highaffinity to their corresponding receptor e.g. epidermal growth factorwhich binds to several receptors including erb-2 which is highlyassociated with tumourgenesis. As described above the fusion constructcan be incorporated into a recombinant viral vector for use in genetherapy e.g. inject patient tissue directly or to transduce patientderived macrophages/moncytes/CD34+ cells ex vivo. The fusion proteinwill be secreted and will bind to tumour cells bearing the erb-2antigen.

Epidermal growth factor (EGF) will bind to its ligand erb-2 (an EGFreceptor) thus obviating the requirement of a ScFv. Erb-2 is highlyassociated with tumour cells (Hynes N E Semin Cancer Biol 1993 February;4(1):19-26, Amplification and over expression of the erbB-2 gene inhuman tumors: its involvement in tumor development, significance as aprognostic factor, and potential as a target for cancer therapy). B7 isfound on the surface of professional antigen presenting cells e.g.macrophages, dendritic cells and B cells. It interacts with it ligandsCD28 and CTL-A4 located on CD4 and CD8 cells. The simultaneousinteraction of B7-CD28/CTL-A4 and MHC-peptide/T cell receptor leads tomassive increase in IL-2 which promotes CD8 (cytotoxic T cell) expansion(Linsley P S, Brady W, Grosmaire L, Aruffo A, Damle N K, Ledbetter J A JExp Med 1991 Mar. 1; 173(3):721-730 Binding of the B cell activationantigen B7 to CD28 costimulates T cell proliferation and interleukin 2mRNA accumulation.) Tumour cells that have been B7 transfected with B7have shown retardation in animal models (Townsend S E, Allison J PScience 1993 15; 259(5093):368-370 Tumor rejection after directcostimulation of CD8+ T cells by B7-transfected melanoma cells). It ishas been reported that B7 will enhance the CTL response to tumourantigens specific to tumour cells thus leading to the destruction of allsuch cells.

Example 15 Production of Cell Lines Expressing Fusion Constructs

The ScFv-IgG gene was excised from pBSII/L/ScFv/hIgG1 by Xho Idigestion, and cloned into pLXSN via the Xho I site, to makepLXSN/ScFv-IgG, such that after chromosomal integration it is undertranscriptional control of the LTR. Virus was made in the human kidneycell line 293T by co-transfecting plasmids containing the MLV gap-polgenes (pCIEGPPD) and the VSV G envelope (pRV67) using the triple plasmidHIT system (Landau & Littman 1992 J Virol 66 5110, Soneoka Y et al 1995NAR 23:628-633). Virus is harvested after 48 hours and used to transduceBHK-21 cells (ATCC# CCL-10). Approximately 24 hours post-transduction,transduced cells are selected by the addition of 1 mg/ml G418 (GibcoBRL) to culture medium. The supernatant from positive colonies washarvested and concentrated by centrifugation through a Centriprep(Amicon, Glos. UK) 10 filter (all proteins larger than 10 kDa arepurified/concentrated) and a Centricon (Amicon) 10 filter. Supernatantswere concentrated approximately 30 fold.

Other fusion proteins are cloned into pLXSN via the Xho I site andexpressed and concentrated using a similar protocol.

FACS Analysis of Fusion Protein Binding with Cells Expressing SpecificLigand

To determine if the ScFv-IgG fusion protein is specific for its antigen,human 5T4, FACS analysis of a human bladder carcinoma tumour line (EJ)or a stable murine cell line expressing h5T4, A9-h5T4 (Myers et al 1994JBC) and a 5T4 negative line A9-neo was carried out. Approximately 5×10⁵A9 or EJ cells, in a round bottom 96 well plate (Falcon) were incubatedwith 100 ul of a 1:5 dilution of concentrated supernatant (as describedabove) for 1 hour at 4oC. After washing, bound protein is detected usingan anti human IgG/FITC conjugated antibody (Dako). Cells were analysedon a Becton Dickinson FACS machine. FACS results show that there is atleast a 1 log shift in fluorescence activity in those 5T4 positive cellstreated with the ScFv-IgG construct compared to the negative controlconstruct consisting of the ScFv protein alone. A9 neo FACS shows thatthere is no non-specific binding of the ScFv component of the fusionprotein.

FACS analysis of ScFv-IgE is carried out similar to above except thatanti-human IgE-FITC (Dako) is used to detect binding of the fusionprotein.

The B7/EGF fusion protein is analysed for binding using FACS andHC11-erb-2 positive cells (Hynes et al 1990). CTLA4-1 g (Ancell, USA) isused to analyse the bioactivity of the B7 component of the bound fusionprotein. Anti-mouse IgG-FITC is used to show CTLA-4 binding.

Example 16 Assembly and Cloning of an ScFv Specific for IL-5

The anti-IL-5 scFv is assembled by RT-PCR using material prepared from ahybridoma line such as the one expressing the humanised Mab to IL-5, SB240563 (Leckie, M J, Am. J. Respir. Crit. Care Med. 159, A624 1999).Techniques are similar to those described by Clackson et al (Geneticallyengineered monoclonal antibodies. Br J Rheumatol. 1991; 30 Suppl2:36-9). Briefly, total RNA is prepared from SB 240563 cells. Firststrand synthesis is performed using MMLV reverse transcriptase usingoligo dT primer. Template cDNAs are amplified by PCR with V_(H) andV_(L) gene specific primer pairs that include restriction enzyme sitesto allow cloning into pKLink, a pBluescript II SK (pBSII) plasmid thatcontains a flexible linker sequence, (Gly₄Ser)₃ (FIG. 5) This forms thesingle chain antibody cDNA (FIG. 6). A double stranded oligonucleotideencoding a translation initiation, Kozak sequence and the human Ig kappalight chain signal peptide for secretion, is then cloned upstream of thescFv.

The whole construct is then excised with Sbf I and Eco RI and clonedinto pONY 8.1 SM (see WO 01/36486) (FIG. 7).

Cloning of scFv Specific for IL-5 into pAdApt

The L-scFv cloned into pBSII is digested with Xba I, filled in to give ablunt end and then digested with Eco RI. The pAdApt vector is digestedwith Hind III filled in to give a blunt end and then digested Eco RI.The two molecules are then ligated to give a recombinant transfervector.

Production of Recombinant Adenovirus Expressing the scFv FusionConstruct

To produce recombinant adenovirus expressing the scFv fusion construct,PerC6 cells are transfected with equimolar amounts of the recombinanttransfer vector containing the fusion construct and an adenovirus Genomevector (AdEasy from Quantum Apligene, Harefield UK). Recombinant virusis then harvested as described in the Crucell protocol.

The recombinant virus can be used as a pharmaceutical composition forthe prevention and/or treatment of asthma.

Example 17 Assembly and Cloning of an scFv Specific for the EnvelopeProtein gp120 of HIV

The anti-HIV scFv is assembled by RT-PCR using material prepared from ahybridoma line expressing a mAb to the envelope protein gp120 of HIV,such as mAb 110.3 (Conelly et al, Virology 295: 554-557, 1994.).Alternatively guided selection is used to make a humanised antibody (seeBeiboer S H et al, J Mol Biol, 2000; 296:833-849) from which the scFv isthen derived. Techniques are similar to that described by Clackson et al(Genetically engineered monoclonal antibodies. Br J. Rheumatol. 1991; 30Suppl 2:36-9). Briefly, Total RNA is prepared from the hybridoma cells.First strand synthesis is performed using MMLV reverse transcriptaseusing oligo dT primer. Template cDNAs are amplified by PCR with V_(H)and V_(L) gene specific primer pairs that include restriction enzymesites, such as those shown below, to allow cloning into pKLink, apBluescript II SK (pBSII) plasmid that contains a flexible linkersequence, (Gly₄Ser)₃ (FIG. 5) This forms the single chain antibodycDNA(FIG. 21). A double stranded oligonucleotide encoding a translationinitiation, Kozak sequence and the human Ig kappa light chain signalpeptide for secretion, is then cloned upstream of the scFv.

The whole construct is then excised with Sbf I and Eco RI and clonedinto pONY 8.1SM (FIG. 8).

Cloning of scFv Specific for the Envelope Protein gp120 of HIV

The L-scFv cloned into pBSII is digested with Xba I, filled in to give ablunt end and then digested with Eco RI. The pAdApt vector is digestedwith Hind III filled in to give a blunt end and then digested Eco RI.The two molecules are then ligated to give a recombinant transfervector.

To produce recombinant adenovirus expressing the scFv fusion constructs,PerC6 cells are transfected with equimolar amounts of the recombinanttransfer vector containing the fusion construct and an adenovirus Genomevector (AdEasy from Quantum Apligene, Harefield UK). Recombinant virusis then harvested as described in the Crucell protocol.

The recombinant virus can be used to enhance a patient's anti-HIVresponse by providing an in vivo factory for a gp120-specific antibody.

Example 18 Anti-TNF Antibodies and Rheumatoid Arthritis

Assembly and Cloning of scFv Specific for Human TNF

The recombinant human anti-TNF antibodies are isolated as described inHoogenboom et al. (Human antibodies that bind human TNF alpha. U.S. Pat.No. 6,090,382) Below is described an example based on the antibody D2E7.Template cDNAs are amplified by PCR with V_(H) and V_(L) gene specificprimer pairs that include restriction enzyme sites, such as those shownbelow, to allow cloning into pKLink, a pBluescript II SK (PBSII) plasmidthat contains a flexible linker sequence, (Gly₄Ser)₃ (FIG. 5) This formsthe single chain antibody cDNA (FIG. 6). A double strandedoligonucleotide encoding a translation initiation, Kozak sequence andthe human Ig kappa light chain signal peptide for secretion, similar tothat described in the construction of the scFv to 5T4 (see WO 01/36486),is then cloned upstream of the scFv (FIG. 6).

The whole construct can then be excised with Sbf I and Eco RI and clonedinto a lentivector such as pONY 8.1 SM (FIG. 9).

In this example the VH is amplified with additional Spe I andMfe/restriction sites at the 5′ end and an additional Age I site at the3′ end. The Spe I and Age I sites are used to clone into pKlink. The VLis amplified with an additional Bam HI restriction site at the 5′ endand an additional Eco RI site at the 3′ end, which are used for cloninginto pKlink.

The leader signal peptide is synthesised as two complementaryoligonucleotides, that are annealed to give restriction enzyme overhangsand then cloned as a double stranded oligonucleotide between the Spe Iand Mfe I sites at the 5′ end of the scFv cDNA.

The Kozak sequence including the ATG start codon (underlined) is in boldand italics.

The V_(H) and V_(L) sequences are shown as SEQ ID NOs 8 and 9respectively. The recombinant virus can be used as a pharmaceuticalcomposition for the prevention and/or treatment of arthritis.

Example 19 Retina Disease

Assembly and cloning of scFv Specific for Human VEGF

The recombinant human anti-VEGF antibodies are isolated as described inVitaliti et a/Cancer Res 2000 Aug. 15; 60(16):4311-4. (Inhibition oftumor angiogenesis by a single-chain antibody directed against vascularendothelial growth factor). Below is described an example based on theantibody sequence available in the GenBank database (submitted by Yan atal; accessin no. AB014341). Template cDNAs are amplified by PCR withV_(H) and V_(L) gene specific primer pairs that include restrictionenzyme sites, such as those shown below, to allow cloning into pKLink, apBluescript II SK (pBSII) plasmid that contains a flexible linkersequence, (Gly₄Ser)₃ (FIG. 5). This forms the single chain antibody cDNA(FIG. 6). A double stranded oligonucleotide encoding a translationinitiation, Kozak sequence and the human Ig kappa light chain signalpeptide for secretion, similar to that described in the construction ofthe scFv to 5T4 (see WO 01/36486), is then cloned upstream of the scFv(FIG. 6).

The whole construct is then be excised with Sbf I and Eco RI and clonedinto a lentivector such as pONY 8.1SM (FIG. 10).

In this example the VH is amplified with additional Spe I and Mfe Irestriction sites at the 5′ end and an additional Age I site at the 3′end. The Spe I and Age/sites are used to clone into pKlink. The VL isamplified with an additional Bam HI restriction site at the 5′ end andan additional Eco RI site at the 3′ end, which are used for cloning intopKlink.

The leader signal peptide is synthesised as two complementaryoligonucleotides, that are annealed to give restriction enzyme overhangsand then cloned as a double stranded oligonucleotide between the Spe Iand Mfe I sites at the 5′ end of the scFv cDNA.

The Kozak sequence including the ATG start codon (underlined) is in boldand italics.

The ScFv sequence is shown as SEQ ID No 10.

The recombinant virus can be used as a pharmaceutical composition forthe prevention and/or treatment of arthritis.

Example 20 Gene Transfer of scFv Proteins In Vivo

The aim of this study was to verify the expression of scFv followingintratumour administration of a viral vector encoding scFv that has beentagged with myc and H is polypeptides.

Experimental Design

An adenoviral vector expressing murine B7.1 fused to scFvmycHis(AdB7-scFv) was used to demonstrate the intra-tumoural delivery of genesthat encode for scFv proteins specific to 5T4. A control adenoviralvector expressing the lac z gene (Adlac z) was also used. Both vectorsused were of the human Ad5 serotype that lacks the genes encoding for E1and E3.

CT26 cells expressing h5T4 (CT26-h5T4) were washed twice in PBS and5×10⁵ cells were injected sc into both flanks of a female Balb/c mouse.Each tumour was established to an average diameter of 5 mm and received3 daily injections of 4×10⁸ pfu Adenovirus in 50 μl. 48 hours after thefinal injection tumours were excised and snap frozen and 100 8 μmsections were cut prior to staining for murine B7.1 or c-myc proteins.

Results

There was no positive staining for c-myc or B7.1 in sections adjacent tothose that were positive for lac z (FIG. 11). This confirms thespecificity of staining by the anti-bodies used and also verifies thatB7.1 positive staining seen is due to gene transfer of the scFv fusionprotein and not the presence of adenovirus.

Gene transfer of B7-scFv was verified on adjacent sections that stainedpositive for B7.1 as well as for the c-myc tag (FIG. 12). Specificity ofstaining was verified by the absence of positive cells in those sectionsthat were incubated in the secondary antibody alone.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

1. A method for delivery of an antibody for treating a non-cancerousdisease in a subject, said method comprising directly administering to acell in the subject a lentiviral vector comprising a nucleotide sequenceencoding an antibody, wherein the antibody is expressed in the cell,thereby stimulating an immune response and treating the non-cancerousdisease in the subject.
 2. The method of claim 1, wherein thenon-cancerous disease is an immune system disease, a cardiovasculardisease, an infectious disease, an eye disease, or a neurologicaldisorder.
 3. The method of claim 1, wherein the antibody is humanized.4. The method of claim 1, wherein the antibody is a monoclonal antibody.5. The method of claim 1, wherein the antibody is a single chainantibody.
 6. The method of claim 1, wherein the antibody is a humanantibody.